Antibodies against BACE1 and use thereof for neural disease immunotherapy

ABSTRACT

The invention provides antagonistic antibodies to BACE1 and methods of using the same for the treatment of neurological disease and disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry pursuant to 35 U.S.C. § 371of International Application No. PCT/US2015/061401, filed Nov. 18, 2015,which claims the benefit of priority of U.S. Provisional Application No.62/081,966, filed Nov. 19, 2014, which is incorporated by referenceherein in its entirety for any purpose.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronicformat. The Sequence Listing is provided as a file entitled“2015-11-16_01146-0040-00PCT_ST25.txt” created on Nov. 16, 2015, whichis 181,893 bytes in size. The information in the electronic format ofthe sequence listing is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates generally to antibodies which are BACE1antagonists that, for example, inhibit or decrease BACE1 activity and tocompositions comprising such antibodies. Additional embodiments includemethods for treating and diagnosing various neurological diseases ordisorders, as well as methods of reducing APP and/or Aβ polypeptides ina patient.

BACKGROUND

Amyloidosis is not a single disease entity but rather a diverse group ofprogressive disease processes characterized by extracellular tissuedeposits of a waxy, starch-like protein called amyloid, whichaccumulates in one or more organs or body systems. As the amyloiddeposits accumulate, they begin to interfere with the normal function ofthe organ or body system. There are at least 15 different types ofamyloidosis. The major forms are primary amyloidosis without knownantecedent, secondary amyloidosis following some other condition, andhereditary amyloidosis.

Many diseases of aging are based on or associated with amyloid-likeproteins and are characterized, in part, by the buildup of extracellulardeposits of amyloid or amyloid-like material that contribute to thepathogenesis, as well as the progression of the disease. These diseasesinclude, but are not limited to, neurological disorders such asAlzheimer's Disease (AD), Lewy body dementia, Down's syndrome,hereditary cerebral hemorrhage with amyloidosis (Dutch type); the GuamParkinson-Dementia complex. Other diseases which are based on orassociated with amyloid-like proteins are progressive supranuclearpalsy, multiple sclerosis, Creutzfeld Jacob disease, Parkinson'sdisease, HIV-related dementia, ALS (amyotropic lateral sclerosis), AdultOnset Diabetes, senile cardiac amyloidosis, endocrine tumors, andothers, including macular degeneration.

The polypeptide β-amyloid (Aβ) is likely to play a central role in thepathogenesis of Alzheimer's disease (AD). Vassar et al., J. Neurosci.29:12787-12794 (2009). Aβ polypeptide accumulation in the CNS results insynaptic dysfunction, axon degeneration and neuronal death. The brainsof AD patients show a characteristic pathology of prominentneuropathologic lesions, such as neurofibrillary tangles (NFTs), andamyloid-rich senile plaques. The major component of amyloid plaques isAβ. These lesions are associated with massive loss of populations ofcentral nervous system (CNS) neurons and their progression accompaniesthe clinical dementia associated with AD.

Aβ is the proteolytic product of the precursor protein, beta amyloidprecursor protein (β-APP or APP). APP is a type-I trans-membrane proteinwhich is sequentially cleaved by two proteases, a β- and γ-secretase.The β-secretase, known asp-site amyloid precursor protein cleavingenzyme 1 (BACE1), first cleaves APP to expose the N-terminus of Aβ,thereby producing a membrane bound fragment known as C99. Vassar et al.,J. Neurosci., 29:12787-12794 (2009) and UniProtKB/Swiss-Prot EntryP56817 (BACE1_HUMAN). The γ-secretase then is able to cleave C99 toproduce the mature Aβ polypeptide. Aβ is produced with heterogenous Ctermini ranging in length from 38 amino acids to 43 amino acids. The 42amino acid form of Aβ (Aβ₄₂) is the fibrillogenic form of Aβ and is overproduced in patients with Down's syndrome and has been suggested to playa role in the early pathogenesis of AD. Vassar et al., J. Neurosci.29:12787-12794 (2009). BACE1 has thus become a therapeutic target as itsinhibition would presumably inhibit APP and Aβ production.

Indeed, BACE1 knock-out mice (BACE1^(−/−)) do not produce cerebral Aβ,confirming that BACE1 is the major, if not only, enzyme responsible forproducing Aβ in the brain. Roberds et al., Human Mol. Genetics10:1317-1324 (2001). Moreover, BACE1 knockout mice in AD models do notform amyloid plaques; cognitive defects and cholinergic dysfunction arerescued as well. McConlogue et al., J. Biol. Chem. 282: 26326-26334(2007); Ohno et al., Neuron 41: 27-33 (2004); and Laird et al., J.Neurosci. 25:11693-11709 (2005). Additionally, BACE1 heterozygousknock-out mice have reduced plaque formation indicating the completeinhibition of BACE1 activity is not necessary for plaque reduction.McConlogue et al., J. Biol. Chem. 282: 26326-26334 (2007).

It would be beneficial to have an effective therapeutic inhibitor ofBACE1 to reduce APP and Aβ production in patients with neurologicaldiseases and disorders, such as AD. The invention provided hereinrelates to such inhibitors, including their use in a variety of methods.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY

The invention provides BACE1 antagonist antibodies and methods of usingthe same. Specifically, the antibodies inhibit or reduce the activity ofBACE1.

In some embodiments, an isolated antibody that binds to BACE1 isprovided, wherein the antibody comprises:

-   -   a) an HVR-H1 sequence selected from SEQ ID NOs: 1 to 6; an        HVR-H2 sequence selected from SEQ ID NOs: 22 to 25; an HVR-H3        sequence selected from SEQ ID NOs: 50 and 51; an HVR-L1 sequence        selected from SEQ ID NOs: 62 and 63; an HVR-L2 sequence selected        from SEQ ID NOs: 69 and 70; and an HVR-L3 sequence selected from        SEQ ID NOs: 75 to 78 and 98; or    -   b) an HVR-H1 sequence selected from SEQ ID NOs: 7 to 21, 218,        and 222 to 224; an HVR-H2 sequence selected from SEQ ID NOs: 26        to 49, 232, 219, and 225; an HVR-H3 sequence selected from SEQ        ID NOs: 52 to 61, 220, 221, 226, and 227; an HVR-L1 sequence        selected from SEQ ID NOs: 64 to 68; an HVR-L2 sequence selected        from SEQ ID NOs: 69 to 74 and 217; and an HVR-L3 sequence        selected from SEQ ID NOs: 79 to 97.

In some embodiments, an isolated antibody that binds to BACE1 isprovided, wherein the antibody comprises the HVR-H1, HVR-H2, HVR-H3,HVR-L1, HVR-L2, and HVR-L3 of an antibody selected from the antibodiesin Table 1. In some embodiments, the antibody is selected from 6266 and6266 variants 1-15. In some embodiments, the antibody comprises anHVR-H1 sequence selected from SEQ ID NOs: 15, 218, and 222 to 224; anHVR-H2 sequence selected from SEQ ID NOs: 29, 219, and 255; an HVR-H3sequence selected from SEQ ID NOs: 52, 220, 221, 226, and 227; an HVR-L1sequence of SEQ ID NO: 65; an HVR-L2 sequence selected from SEQ ID NOs:71, 73, and 217; and an HVR-L3 sequence of SEQ ID NO: 80. In someembodiments, the antibody comprises:

-   -   a) a heavy chain variable domain sequence having at least 90%        sequence identity to an amino acid sequence selected from SEQ ID        NOs: 99 to 147, 194 to 200, and 209 to 216; or    -   b) a light chain variable domain sequence having at least 90%        sequence identity to an amino acid sequence selected from SEQ ID        NOs: 148 to 178, 187 to 194, and 201 to 208; or    -   c) a heavy chain variable domain sequence as in (a) and a light        chain variable domain sequence as in (b).

In some embodiments, an isolated antibody that binds BACE1 comprises:

-   -   a) a heavy chain variable domain sequence selected from SEQ ID        NOs: 99 to 147, 194 to 200, and 209 to 216; or    -   b) a light chain variable domain sequence selected from SEQ ID        NOs: 148 to 178, 187 to 194, and 201 to 208; or    -   c) a heavy chain variable domain sequence as in (a) and a light        chain variable domain sequence as in (b).

In some embodiments, an isolated antibody that binds BACE1 comprises:

-   -   a) a heavy chain variable domain sequence having at least 90%        sequence identity to a sequence selected from SEQ ID NOs: 138,        194 to 200, and 209 to 216; or    -   b) a light chain variable domain sequence having at least 90%        sequence identity to a sequence selected from SEQ ID NOs: 156,        187 to 194, and 201 to 208; or    -   c) a heavy chain variable domain sequence as in (a) and a light        chain variable domain sequence as in (b).

In some embodiments, an isolated antibody that binds BACE1 comprises:

-   -   a) a heavy chain variable domain sequence selected from SEQ ID        NOs: 138, 194 to 200, and 209 to 216; or    -   b) a light chain variable domain sequence selected from SEQ ID        NOs: 156, 187 to 194, and 201 to 208; or    -   c) a heavy chain variable domain sequence as in (a) and a light        chain variable domain sequence as in (b); or    -   d) a heavy chain variable domain sequence and a light chain        variable domain sequence of an antibody selected from 6266 and        6266 variants 1-15.

In some embodiments, the isolated antibody modulates the activity ofBACE1. In some embodiments, the antibody inhibits the activity of BACE1.In some embodiments, BACE1 activity is measured using a homogeneoustime-resolved fluorescence (HTRF) assay. In some embodiments, BACE1activity is measured using a cell line that expresses a BACE1 substrate.In some embodiments, the BACE1 substrate is amyloid precursor protein(APP). In some embodiments, BACE1 activity is measured in tissue from ananimal that has been administered the anti-BACE1 antibody. In someembodiments, the tissue is brain tissue. In some embodiments, the animalis selected from a mouse, rat, rabbit, dog, monkey, and non-humanprimate.

In some embodiments, the antibody is an allosteric inhibitor of BACE1activity. In some embodiments, the antibody binds BACE1 with an affinity(KD) of between 0.1 nM and 10 nM, or between 0.1 nM and 8 nM, or between0.1 nM and 7 nM, or between 0.1 nM and 5 nM, or between 0.5 nM and 5 nM,or between 0.1 nM and 3 nM, or between 0.5 nM and 3 nM, as measured bysurface plasmon resonance (SPR). In some embodiments, the antibodyachieves a maximum inhibition of BACE1 activity of greater than 60%,greater than 70%, greater than 75%, or greater than 80%, as measured,for example, using the dissociated cortical neuron culture assay.

An antibody of the invention can be in any number of forms. For example,an antibody of the invention can be a human antibody or chimericantibody. In other aspects the antibody of the invention is a fulllength antibody or a fragment thereof (e.g., a fragment comprising anantigen binding component). In some embodiments, the antibody fragmentis selected from a Fab, Fab′, Fab′-SH, F(ab′)₂, Fv, and scFv. In someembodiments, the antibody is a full length IgG1 antibody. In otheraspects of the invention, the antibody is a monoclonal antibody. Inanother aspect, an antibody of the invention can be linked or conjugatedto an agent or moiety, e.g. a cytotoxic agent, to create animmunoconjugate.

In some embodiments, a pharmaceutical formulation is provided whichcomprises an antibody of the invention and a pharmaceutically acceptablecarrier. In additional embodiments an isolated nucleic acid encoding anantibody of the invention is provided, as well as vector that comprisesthe nucleic acid encoding an antibody of the invention. In anotheraspect, a host cell comprising the nucleic acid encoding an antibody ofthe invention is provided as well as methods for producing an antibodyof the invention comprising culturing the host cell comprising thenucleic acid encoding an antibody of the invention under conditionssuitable for production of the antibody.

In another embodiment, a method of treating an individual having aneurological disease or disorder comprising administering to theindividual an effective amount of an antibody of the invention isprovided.

In an additional embodiment, a method of reducing amyloid plaques, orinhibiting amyloid plaque formation, in a patient suffering from, or atrisk of contracting, a neurological disease or disorder comprisingadministering to the individual an effective amount of an antibody ofthe invention is provided.

In some embodiments, a method of reducing Aβ protein in a patientcomprising administering to the patient an effective amount of anantibody of the invention. In some aspects, the patient is sufferingfrom, or at risk of contracting, a neurological disease or disorder.

In another embodiment, a method of inhibiting axon degeneration in apatient comprising administering to the patient an effective amount ofan antibody of the invention is provided.

In an additional embodiment, a method of diagnosing a neurologicaldisease or disorder in patient comprising contacting a biological sampleisolated from the patient with an antibody of the invention underconditions suitable for binding of the antibody to a BACE1 polypeptide,and detecting whether a complex is formed between the antibody and theBACE1 polypeptide.

In some embodiments, a method of determining whether a patient iseligible for therapy with an anti-BACE1 antibody, comprising contactinga biological sample isolated from the patient with an antibody of theinvention under conditions suitable for binding of the antibody to aBACE1 polypeptide, and detecting whether a complex is formed between theantibody and the BACE1 polypeptide, wherein the presence of a complexbetween the antibody and BACE1 is indicative of a patient eligible fortherapy with an anti-BACE1 antibody. In some aspects the patient issuffering from, or at risk of contracting, a neurological disease ordisorder.

In some aspects, biological samples that may be used in the diagnosis ofa neurological disease or condition; or for predicting responsiveness,or determining eligibility, of a patient to a treatment with a BACE1antibody include, but are not limited to, fluids such as serum, plasma,saliva, gastric secretions, mucus, cerebrospinal fluid, lymphatic fluidand the like or tissue or cell samples obtained from an organism such asneuronal, brain, cardiac or vascular tissue.

In some aspects of the methods of the invention, the patient ismammalian. In another aspect, the patient is human. In another aspect,the neurological disease or disorder is selected from the groupconsisting of Alzheimer's disease (AD), traumatic brain injury, stroke,glaucoma, dementia, muscular dystrophy (MD), multiple sclerosis (MS),amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman'ssyndrome, Liddle syndrome, Paget's disease, traumatic brain injury, Lewybody disease, postpoliomyelitis syndrome, Shy-Draeger syndrome,olivopontocerebellar atrophy, Parkinson's disease, multiple systematrophy, striatonigral degeneration, supranuclear palsy, bovinespongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru,Gerstmann-Straussler-Scheinker disease, chronic wasting disease, fatalfamilial insomnia, bulbar palsy, motor neuron disease, Canavan disease,Huntington's disease, neuronal ceroid-lipofuscinosis, Alexander'sdisease, Tourette's syndrome, Menkes kinky hair syndrome, Cockaynesyndrome, Halervorden-Spatz syndrome, lafora disease, Rett syndrome,hepatolenticular degeneration, Lesch-Nyhan syndrome, andUnverricht-Lundborg syndrome, dementia (including, but not limited to,Pick's disease, and spinocerebellar ataxia). In some aspects, theneurological disease or disorder is Alzheimer's disease. In someembodiments, the neurological disease or disorder is selected from thegroup consisting of Alzheimer's disease, stroke, traumatic brain injuryand glaucoma.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D show the epitope bin and heavy chain HVR-H1, HVR-H2, andHVR-H3 sequences of certain anti-BACE1 antibodies described herein.

FIGS. 2A-2C show the epitope bin and light chain HVR-L1, HVR-L2, andHVR-L3 sequences of certain anti-BACE1 antibodies described herein.

FIGS. 3A-3G show the epitope bin and heavy chain variable region (VH)sequences of certain anti-BACE1 antibodies described herein.

FIGS. 4A-4F show the epitope bin and light chain variable region (VL)sequences of certain anti-BACE1 antibodies described herein.

FIGS. 5A-5D show the affinity (KD) of certain anti-BACE1 antibodies forhuman BACE1 at pH 7.5 (column 2), murine BACE1 at pH 7.5 (column 3),human BACE1 at pH 5.0 (column 4), and murine BACE1 at pH 5.0 (column 5)using an Octet® system (ForteBio); and the affinity (K_(D)) of certainanti-BACE1 antibodies for human BACE1 by surface plasmon resonance(Biacore™). For certain antibodies, affinities deteremined in twoseparate assays are shown.

FIG. 6 shows modulation of BACE1 activity by the anti-BACE1 antibodiesusing a short substrate assay.

FIGS. 7A-7B shows modulation of BACE1 activity by the anti-BACE1antibodies using a long substrate assay and a short substrate assay.

FIGS. 8A-8C show in vitro modulation of APP processing in cells by theanti-BACE1 antibodies.

FIG. 9 shows the effects of the indicated anti-BACE1 antibodies onprocessing of endogenous amyloid precursor protein (APP). Experimentswere performed using cultures of E16.5 cortical neurons from wild-typeCD1 mice.

FIG. 10 shows Aβ_(x-40) levels observed in the brain (cortex) of micetreated with 100 mg/kg of the indicated anti-BACE1 antibodies or controlIgG antibody.

FIG. 11 shows serum antibody concentration over time in cynomolgusmonkeys following a single IV dose.

FIG. 12A-B show the (A) light chain variable region sequences and (B)heavy chain variable region sequences of the affinity-matured variants1-7 of antibody 6266.

FIG. 13A-B show the (A) light chain variable region sequences and (B)heavy chain variable region sequences of the affinity-matured variants8-15 of antibody 6266.

FIG. 14 shows affinity constants and melting temperature for theaffinity-matured variants of antibody 6266.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The terms “anti-beta-secretase antibody”, “anti-BACE1 antibody”, “anantibody that binds to beta-secretase” and “an antibody that binds toBACE1” refer to an antibody that is capable of binding BACE1 withsufficient affinity such that the antibody is useful as a diagnosticand/or therapeutic agent in targeting BACE1. In some embodiments, theextent of binding of an anti-BACE1 antibody to an unrelated, non-BACE1protein is less than about 10% of the binding of the antibody to BACE1as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments,an antibody that binds to BACE1 has a dissociation constant (Kd) of ≤1μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ Mor less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). Incertain embodiments, an anti-BACE1 antibody binds to an epitope of BACE1that is conserved among BACE1 from different species and isoforms.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹¹³, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In some embodiments, a human IgG heavy chain Fcregion extends from Cys226, or from Pro230, to the carboxyl-terminus ofthe heavy chain. However, the C-terminal lysine (Lys447) of the Fcregion may or may not be present. Unless otherwise specified herein,numbering of amino acid residues in the Fc region or constant region isaccording to the EU numbering system, also called the EU index, asdescribed in Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md., 1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. Insome embodiments, for the VL, the subgroup is subgroup kappa I as inKabat et al., supra. In some embodiments, for the VH, the subgroup issubgroup III as in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) With the exception of CDR1in VH, CDRs generally comprise the amino acid residues that form thehypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro andFransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-BACE1 antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “BACE1,” as used herein, refers to any native beta-secretase 1(also called β-site amyloid precursor protein cleaving enzyme 1,membrane-associated aspartic protease 2, memapsin 2, aspartyl protease 2or Asp2) from any vertebrate source, including mammals such as primates(e.g. humans) and rodents (e.g., mice and rats), unless otherwiseindicated. The term encompasses “full-length,” unprocessed BACE1 as wellas any form of BACE1 that results from processing in the cell. The termalso encompasses naturally occurring variants of BACE1, e.g., splicevariants or allelic variants. The amino acid sequence of an exemplaryBACE1 polypeptide is shown in SEQ ID NO:179 below, and is the sequencefor human BACE1, isoform A as reported in Vassar et al., Science286:735-741 (1999), which is incorporated herein by reference in itsentirety.

(SEQ ID NO: 179) MAQALPWLLLWMGAGVLPAHGTQHGIRLPLRSGLGGAPLGLRLPRETDEEPEEPGRRGSFVEMVDNLRGKSGQGYYVEMTVGSPPQTLNILVDTGSSNFAVGAAPHPFLHRYYQRQLSSTYRDLRKGVYVPYTQGKWEGELGTDLVSIPHGPNVTVRANIAAITESDKFFINGSNWEGILGLAYAEIARPDDSLEPFFDSLVKQTHVPNLFSLQLCGAGFPLNQSEVLASVGGSMIIGGIDHSLYTGSLWYTPIRREWYYEVIIVRVEINGQDLKMDCKEYNYDKSIVDSGTTNLRLPKKVFEAAVKSIKAASSTEKFPDGFWLGEQLVCWQAGTTPWNIFPVISLYLMGEVTNQSFRITILPQQYLRPVEDVATSQDDCYKFAISQSSTGTVMGAVIMEGFYVVFDRARKRIGFAVSACHVHDEFRTAAVEGPFVTLDMEDCGYNIPQTDESTLMTIAYVMAAICALFMLPLCLMVCQWCCLRCLRQQHDDFADDISLLK

Several other isoforms of human BACE1 exist including isoforms B, C andD. See UniProtKB/Swiss-Prot Entry P56817, which is incorporated hereinby reference in its entirety. Isoform B is shown in SEQ ID NO:180 anddiffers from isoform A (SEQ ID NO:179) in that it is missing amino acids190-214 (i.e. deletion of amino acids 190-214 of SEQ ID NO:179). IsoformC is shown in SEQ ID NO:181 and differs from isoform A (SEQ ID NO:179)in that it is missing amino acids 146-189 (i.e. deletion of amino acids146-189 of (SEQ ID NO:179). Isoform D is shown in SEQ ID NO:182 anddiffers from isoform A (SEQ ID NO:179) in that it is missing amino acids146-189 and 190-214 (i.e. deletion of amino acids 146-189 and 190-214 ofSEQ ID NO:179).

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W. H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

The terms “neurological disorder” or “neurological disease” refer to ordescribe a disease or disorder of the central and/or peripheral nervoussystem in mammals. Examples of neurological disorders include, but arenot limited to the following list of disease and disorders. Neuropathydisorders are diseases or abnormalities of the nervous systemcharacterized by inappropriate or uncontrolled nerve signaling or lackthereof, and include, but are not limited to, chronic pain (includingnociceptive pain (pain caused by an injury to body tissues, includingcancer-related pain), neuropathic pain (pain caused by abnormalities inthe nerves, spinal cord, or brain), and psychogenic pain (entirely ormostly related to a psychological disorder), headache, migraine,neuropathy, and symptoms and syndromes often accompanying suchneuropathy disorders such as vertigo or nausea. Amyloidoses are a groupof diseases and disorders associated with extracellular proteinaceousdeposits in the CNS, including, but not limited to, secondaryamyloidosis, age-related amyloidosis, Alzheimer's Disease (AD), mildcognitive impairment (MCI), Lewy body dementia, Down's syndrome,hereditary cerebral hemorrhage with amyloidosis (Dutch type); the GuamParkinson-Dementia complex, cerebral amyloid angiopathy, Huntington'sdisease, progressive supranuclear palsy, multiple sclerosis; CreutzfeldJacob disease, Parkinson's disease, transmissible spongiformencephalopathy, HIV-related dementia, amyotropic lateral sclerosis(ALS), inclusion-body myositis (IBM), and ocular diseases relating tobeta-amyloid deposition (i.e., macular degeneration, drusen-relatedoptic neuropathy, and cataract). Cancers of the CNS are characterized byaberrant proliferation of one or more CNS cell (i.e., a neural cell) andinclude, but are not limited to, glioma, glioblastoma multiforme,meningioma, astrocytoma, acoustic neuroma, chondroma, oligodendroglioma,medulloblastomas, ganglioglioma, Schwannoma, neurofibroma,neuroblastoma, and extradural, intramedullary or intradural tumors.Ocular diseases or disorders are diseases or disorders of the eye, whichfor the purposes herein is considered a CNS organ subject to the BBB.Ocular diseases or disorders include, but are not limited to, disordersof sclera, cornea, iris and ciliary body (i.e., scleritis, keratitis,corneal ulcer, corneal abrasion, snow blindness, arc eye, Thygeson'ssuperficial punctate keratopathy, corneal neovascularisation, Fuchs'dystrophy, keratoconus, keratoconjunctivitis sicca, iritis and uveitis),disorders of the lens (i.e., cataract), disorders of choroid and retina(i.e., retinal detachment, retinoschisis, hypertensive retinopathy,diabetic retinopathy, retinopathy, retinopathy of prematurity,age-related macular degeneration, macular degeneration (wet or dry),epiretinal membrane, retinitis pigmentosa and macular edema), glaucoma,floaters, disorders of optic nerve and visual pathways (i.e., Leber'shereditary optic neuropathy and optic disc drusen), disorders of ocularmuscles/binocular movement accommodation/refraction (i.e., strabismus,ophthalmoparesis, progressive external opthalmoplegia, esotropia,exotropia, hypermetropia, myopia, astigmatism, anisometropia, presbyopiaand ophthalmoplegia), visual disturbances and blindness (i.e.,amblyopia, Lever's congenital amaurosis, scotoma, color blindness,achromatopsia, nyctalopia, blindness, river blindness andmicro-opthalmia/coloboma), red eye, Argyll Robertson pupil,keratomycosis, xerophthalmia and andaniridia. Viral or microbialinfections of the CNS include, but are not limited to, infections byviruses (i.e., influenza, HIV, poliovirus, rubella), bacteria (i.e.,Neisseria sp., Streptococcus sp., Pseudomonas sp., Proteus sp., E. coli,S. aureus, Pneumococcus sp., Meningococcus sp., Haemophilus sp., andMycobacterium tuberculosis) and other microorganisms such as fungi(i.e., yeast, Cryptococcus neoformans), parasites (i.e., Toxoplasmagondii) or amoebas resulting in CNS pathophysiologies including, but notlimited to, meningitis, encephalitis, myelitis, vasculitis and abscess,which can be acute or chronic. Inflammation of the CNS is inflammationthat is caused by an injury to the CNS, which can be a physical injury(i.e., due to accident, surgery, brain trauma, spinal cord injury,concussion) or an injury due to or related to one or more other diseasesor disorders of the CNS (i.e., abscess, cancer, viral or microbialinfection). Ischemia of the CNS, as used herein, refers to a group ofdisorders relating to aberrant blood flow or vascular behavior in thebrain or the causes therefor, and includes, but is not limited to, focalbrain ischemia, global brain ischemia, stroke (i.e., subarachnoidhemorrhage and intracerebral hemorrhage), and aneurysm.Neurodegenerative diseases are a group of diseases and disordersassociated with neural cell loss of function or death in the CNS, andinclude, but are not limited to, adrenoleukodystrophy, Alexander'sdisease, Alper's disease, amyotrophic lateral sclerosis, ataxiatelangiectasia, Batten disease, cockayne syndrome, corticobasaldegeneration, degeneration caused by or associated with an amyloidosis,Friedreich's ataxia, frontotemporal lobar degeneration, Kennedy'sdisease, multiple system atrophy, multiple sclerosis, primary lateralsclerosis, progressive supranuclear palsy, spinal muscular atrophy,transverse myelitis, Refsum's disease, and spinocerebellar ataxia.Seizure diseases and disorders of the CNS involve inappropriate and/orabnormal electrical conduction in the CNS, and include, but are notlimited to, epilepsy (i.e., absence seizures, atonic seizures, benignRolandic epilepsy, childhood absence, clonic seizures, complex partialseizures, frontal lobe epilepsy, febrile seizures, infantile spasms,juvenile myoclonic epilepsy, juvenile absence epilepsy, Lennox-Gastautsyndrome, Landau-Kleffner Syndrome, Dravet's syndrome, Otahara syndrome,West syndrome, myoclonic seizures, mitochondrial disorders, progressivemyoclonic epilepsies, psychogenic seizures, reflex epilepsy, Rasmussen'sSyndrome, simple partial seizures, secondarily generalized seizures,temporal lobe epilepsy, toniclonic seizures, tonic seizures, psychomotorseizures, limbic epilepsy, partial-onset seizures, generalized-onsetseizures, status epilepticus, abdominal epilepsy, akinetic seizures,autonomic seizures, massive bilateral myoclonus, catamenial epilepsy,drop seizures, emotional seizures, focal seizures, gelastic seizures,Jacksonian March, Lafora Disease, motor seizures, multifocal seizures,nocturnal seizures, photosensitive seizure, pseudo seizures, sensoryseizures, subtle seizures, sylvan seizures, withdrawal seizures, andvisual reflex seizures) Behavioral disorders are disorders of the CNScharacterized by aberrant behavior on the part of the afflicted subjectand include, but are not limited to, sleep disorders (i.e., insomnia,parasomnias, night terrors, circadian rhythm sleep disorders, andnarcolepsy), mood disorders (i.e., depression, suicidal depression,anxiety, chronic affective disorders, phobias, panic attacks,obsessive-compulsive disorder, attention deficit hyperactivity disorder(ADHD), attention deficit disorder (ADD), chronic fatigue syndrome,agoraphobia, post-traumatic stress disorder, bipolar disorder), eatingdisorders (i.e., anorexia or bulimia), psychoses, developmentalbehavioral disorders (i.e., autism, Rett's syndrome, Aspberger'ssyndrome), personality disorders and psychotic disorders (i.e.,schizophrenia, delusional disorder, and the like). Lysosomal storagedisorders are metabolic disorders which are in some cases associatedwith the CNS or have CNS-specific symptoms; such disorders include, butare not limited to Tay-Sachs disease, Gaucher's disease, Fabry disease,mucopolysaccharidosis (types I, II, III, IV, V, VI and VII), glycogenstorage disease, GM1-gangliosidosis, metachromatic leukodystrophy,Farber's disease, Canavan's leukodystrophy, and neuronal ceroidlipofuscinoses types 1 and 2, Niemann-Pick disease, Pompe disease, andKrabbe's disease.

II. Compositions and Methods

In some aspects, the invention is based, in part, on antibodies whichbind BACE1 and reduce and/or inhibit BACE1 activity. In certainembodiments, antibodies that bind to the active site or an exosite ofBACE1 are provided.

A. Exemplary Anti-BACE1 Antibodies

In some embodiments, anti-BACE1 antibodies are provided. In someembodiments, an anti-BACE1 antibody provided herein is an allostericinhibitor of BACE1 activity. Nonlimiting exemplary anti-BACE1 antibodiesinclude antibodies comprising the heavy chain and light chain variableregions of the antibodies listed in Table 1. The heavy and light chainvariable regions of the antibodies listed in Table 1 are shown in FIGS.3 and 4, respectively.

TABLE 1 Anti-BACE1 Antibodies Ab Ab Ab Ab Ab Ab 5531 5572 5893 6290 63115987 5586 5536 5887 6291 6309 6303 5583 5571 6275 6293 6310 6266 55325883 6279 6289 6308 6271 5592 5890 6276 5747 5990 6297 5878 5891 58885982 6307 6294 5874 5892 5894 5985 5931 5932 5875 5884 5543 5983 62986313 5876 6272 5643 5984 5930 6314 5880 6270 5644 5986 5988 6315 58816273 5896 6296 6299 5933 5260 6274 5902 5897 6300 6285 6288 5539 59035905 6305 6280 6266.1 6266.2 6266.3 6266.4 6266.5 6266.6 6266.7 6266.86266.9 6266.10 6266.11 6266.12 6266.13 6266.14 6266.15

In some embodiments, an anti-BACE1 antibody described herein, includingbut not limited to antibodies comprising one or more HVRs, or all sixHVRs, of an antibody listed in Table 1, is an allosteric inhibitor ofBACE1 activity. In some embodiments, an anti-BACE1 antibody binds BACE1with an affinity (KD) of less than 10 nM, less than 9 nM, less than 8nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, orless than 3 nM, as measured by surface plasmon resonance (SPR). In someembodiments, an anti-BACE1 antibody binds BACE1 with an affinity (KD) ofbetween 0.1 nM and 10 nM, or between 0.1 nM and 8 nM, or between 0.1 nMand 7 nM, or between 0.1 nM and 5 nM, or between 0.5 nM and 5 nM, orbetween 0.1 nM and 3 nM, or between 0.5 nM and 3 nM, as measured bysurface plasmon resonance (SPR). In some embodiments, an anti-BACE1antibody achieves a maximum inhibition of BACE1 activity of greater than60%, greater than 70%, greater than 75%, or greater than 80%, asmeasured, for example, using the dissociated cortical neuron cultureassay described in Example 2E.

In some aspects, the invention provides an anti-BACE1 antibodycomprising at least one, two, three, four, five, or six HVRs of anantibody selected from the anti-BACE1 antibodies listed in Table 1.FIGS. 1 and 2 show the heavy chain and light chain HVR sequences,respectively, of each of those antibodies. In some embodiments, theinvention provides an anti-BACE1 antibody comprising HVR-H1, HVR-H2,HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of an antibody selected from theanti-BACE1 antibodies listed in Table 1.

In some embodiments, an anti-BACE1 antibody is provided, wherein theantibody comprises an HVR-H1 sequence selected from SEQ ID NOs: 1 to 6;an HVR-H2 sequence selected from SEQ ID NOs: 22 to 25; an HVR-H3sequence selected from SEQ ID NOs: 50 and 51; an HVR-L1 sequenceselected from SEQ ID NOs: 62 and 63; an HVR-L2 sequence selected fromSEQ ID NOs: 69 and 70; and an HVR-L3 sequence selected from SEQ ID NOs:75 to 78 and 98. In some embodiments, an anti-BACE1 antibody isprovided, wherein the antibody comprises an HVR-H1 sequence selectedfrom SEQ ID NOs: 7 to 21, 218, and 222 to 224; an HVR-H2 sequenceselected from SEQ ID NOs: 26 to 49, 232, 219, and 225; an HVR-H3sequence selected from SEQ ID NOs: 52 to 61, 220, 221, 226, and 227; anHVR-L1 sequence selected from SEQ ID NOs: 64 to 68; an HVR-L2 sequenceselected from SEQ ID NOs: 69 to 74 and 217; and an HVR-L3 sequenceselected from SEQ ID NOs: 79 to 97. In some embodiments, an anti-BACE1antibody is provided, wherein the antibody comprises an HVR-H1 sequenceselected from SEQ ID NOs: 15, 218, and 222 to 224; an HVR-H2 sequenceselected from SEQ ID NOs: 29, 219, and 255; an HVR-H3 sequence selectedfrom SEQ ID NOs: 52, 220, 221, 226, and 227; an HVR-L1 sequence of SEQID NO: 65; an HVR-L2 sequence selected from SEQ ID NOs: 71, 73, and 217;and an HVR-L3 sequence of SEQ ID NO: 80. In some embodiments, ananti-BACE1 antibody is provided, wherein the antibody comprises anHVR-H1 of SEQ ID NO: 15; an HVR-H2 sequence of SEQ ID NO: 29; an HVR-H3sequence of SEQ ID NO: 52; an HVR-L1 sequence of SEQ ID NO: 65; anHVR-L2 sequence of SEQ ID NO: 71; and an HVR-L3 sequence of SEQ ID NO:80. In some embodiments, an anti-BACE1 antibody is provided, wherein theantibody comprises an HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3of an antibody selected from antibody 6266 variants 1-15.

In some aspects, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected of an antibodyselected from the anti-BACE1 antibodies listed in Table 1. In someembodiments, the invention provides an antibody comprising HVR-H1,HVR-H2, and HVR-H3 of an antibody selected from the anti-BACE1antibodies listed in Table 1. In some embodiments, an anti-BACE1antibody is provided, wherein the antibody comprises an HVR-H1 sequenceselected from SEQ ID NOs: 1 to 6; an HVR-H2 sequence selected from SEQID NOs: 22 to 25; and an HVR-H3 sequence selected from SEQ ID NOs: 50and 51. In some embodiments, an anti-BACE1 antibody is provided, whereinthe antibody comprises an HVR-H1 sequence selected from SEQ ID NOs: 7 to21, 218, and 222 to 224; and HVR-H2 sequence selected from SEQ ID NOs:26 to 49, 232, 219, and 225; and an HVR-H3 sequence selected from SEQ IDNOs: 52 to 61, 220, 221, 226, and 227. In some embodiments, ananti-BACE1 antibody is provided, wherein the antibody comprises anHVR-H1 sequence selected from SEQ ID NOs: 15, 218, and 222 to 224; anHVR-H2 sequence selected from SEQ ID NOs: 29, 219, and 255; and anHVR-H3 sequence selected from SEQ ID NOs: 52, 220, 221, 226, and 227. Insome embodiments, an anti-BACE1 antibody is provided, wherein theantibody comprises an HVR-H1 of SEQ ID NO: 15; an HVR-H2 sequence of SEQID NO: 29; and an HVR-H3 sequence of SEQ ID NO: 52. In some embodiments,an anti-BACE1 antibody is provided, wherein the antibody comprises anHVR-H1, HVR-H2, and HVR-H3 of an antibody selected from antibody 6266variants 1-15.

In some aspects, the invention provides an antibody comprising at leastone, at least two, or all three VL HVR sequences selected of an antibodyselected from the anti-BACE1 antibodies listed in Table 1. In someembodiments, the invention provides an antibody comprising HVR-L1,HVR-L2, and HVR-L3 of an antibody selected from the anti-BACE1antibodies listed in Table 1. In some embodiments, an anti-BACE1antibody is provided, wherein the antibody comprises an HVR-L1 sequenceselected from SEQ ID NOs: 62 and 63; an HVR-L2 sequence selected fromSEQ ID NOs: 69 and 70; and an HVR-L3 sequence selected from SEQ ID NOs:75 to 78 and 98. In some embodiments, an anti-BACE1 antibody isprovided, wherein the antibody comprises an HVR-L1 sequence selectedfrom SEQ ID NOs: 64 to 68; an HVR-L2 sequence selected from SEQ ID NOs:69 to 74 and 217; and an HVR-L3 sequence selected from SEQ ID NOs: 79 to97. In some embodiments, an anti-BACE1 antibody is provided, wherein theantibody comprises an HVR-L1 sequence of SEQ ID NO: 65; an HVR-L2sequence of SEQ ID NO: 71, 73, or 217; and an HVR-L3 sequence of SEQ IDNO: 80. In some embodiments, an anti-BACE1 antibody is provided, whereinthe antibody comprises an HVR-L1 sequence of SEQ ID NO: 65; an HVR-L2sequence of SEQ ID NO: 71; and an HVR-L3 sequence of SEQ ID NO: 80. Insome embodiments, an anti-BACE1 antibody is provided, wherein theantibody comprises an HVR-L1, HVR-L2, and HVR-L3 of an antibody selectedfrom antibody 6266 variants 1-15.

In another aspect, a heavy chain is provided, comprising a VH domaincomprising at least one, at least two, or all three VH HVR sequences ofan antibody selected from the anti-BACE1 antibodies listed in Table 1.In some embodiments, a heavy chain is provided, comprising a VH domaincomprising all three VH HVR sequences of an antibody selected from theanti-BACE1 antibodies listed in Table 1. In another aspect, a heavychain is provided, comprising a VH domain comprising an HVR-H1 sequenceselected from SEQ ID NOs: 1 to 6; an HVR-H2 sequence selected from SEQID NOs: 22 to 25; and an HVR-H3 sequence selected from SEQ ID NOs: 50and 51. In another aspect, a heavy chain is provided, comprising a VHdomain comprising an HVR-H1 sequence selected from SEQ ID NOs: 7 to 21,218, and 222 to 224; and HVR-H2 sequence selected from SEQ ID NOs: 26 to49, 232, 219, and 225; and an HVR-H3 sequence selected from SEQ ID NOs:52 to 61, 220, 221, 226, and 227. In some embodiments, a heavy chain isprovided, comprising a VH domain comprising an HVR-H1 sequence selectedfrom SEQ ID NOs: 15, 218, and 222 to 224; an HVR-H2 sequence selectedfrom SEQ ID NOs: 29, 219, and 255; and an HVR-H3 sequence selected fromSEQ ID NOs: 52, 220, 221, 226, and 227. In some embodiments, a heavychain is provided, comprising a VH domain comprising an HVR-H1 of SEQ IDNO: 15; an HVR-H2 sequence of SEQ ID NO: 29; and an HVR-H3 sequence ofSEQ ID NO: 52. In some embodiments, a heavy chain is provided,comprising a VH domain comprising an HVR-H1, HVR-H2, and HVR-H3 of anantibody selected from antibody 6266 variants 1-15.

In another aspect, a light chain is provided, comprising a VL domaincomprising at least one, at least two, or all three VL HVR sequences ofan antibody selected from the anti-BACE1 antibodies listed in Table 1.In some embodiments, a light chain is provided, comprising a VL domaincomprising all three VL HVR sequences of an antibody selected from theanti-BACE1 antibodies listed in Table 1. In another aspect, a lightchain is provided, comprising a VL domain comprising an HVR-L1 sequenceselected from SEQ ID NOs: 62 and 63; an HVR-L2 sequence selected fromSEQ ID NOs: 69 and 70; and an HVR-L3 sequence selected from SEQ ID NOs:75 to 78 and 98. In another aspect, a light chain is provided,comprising a VL domain comprising an HVR-L1 sequence selected from SEQID NOs: 64 to 68; an HVR-L2 sequence selected from SEQ ID NOs: 69 to 74and 217; and an HVR-L3 sequence selected from SEQ ID NOs: 79 to 97. Insome embodiments, a light chain is provided, comprising a VL domaincomprising an HVR-L1 sequence of SEQ ID NO: 65; an HVR-L2 sequence ofSEQ ID NO: 71, 73, or 217; and an HVR-L3 sequence of SEQ ID NO: 80. Insome embodiments, a light chain is provided, comprising a VL domaincomprising an HVR-L1 sequence of SEQ ID NO: 65; an HVR-L2 sequence ofSEQ ID NO: 71; and an HVR-L3 sequence of SEQ ID NO: 80. In someembodiments, a light chain is provided, comprising a VL domaincomprising an HVR-L1, HVR-L2, and HVR-L3 of an antibody selected fromantibody 6266 variants 1-15.

In another aspect, an anti-BACE1 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH of ananti-BACE1 antibody of Table 1. In some embodiments, an anti-BACE1antibody comprises a heavy chain variable domain (VH) sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to an amino acid sequence selected from SEQ ID NOs: 99 to 147,194 to 200, and 209 to 216. In some embodiments, an anti-BACE1 antibodycomprises a heavy chain variable domain (VH) sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence selected from SEQ ID NOs: 138, 194to 200, and 209 to 216. In some embodiments, an anti-BACE1 antibodycomprises a heavy chain variable domain (VH) sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 138. In certainembodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-BACE1 antibody comprising that sequenceretains the ability to bind to BACE1 and/or inhibit or reduce BACE1activity. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in any one of SEQ ID NO: 99 to147. In certain embodiments, substitutions, insertions, or deletionsoccur in regions outside the HVRs (i.e., in the FRs). Optionally, theanti-BACE1 antibody comprises the VH sequence in any one of SEQ ID NO:99 to 147, 194 to 200, and 209 to 216, including post-translationalmodifications of that sequence. In a particular embodiment, the VHcomprises one, two or three HVRs of an anti-BACE1 antibody listed inTable 1. In some embodiments, the VH comprises an HVR-H1 sequenceselected from SEQ ID NOs: 1 to 6; an HVR-H2 sequence selected from SEQID NOs: 22 to 25; and an HVR-H3 sequence selected from SEQ ID NOs: 50and 51. In some embodiments, the VH comprises an HVR-H1 sequenceselected from SEQ ID NOs: 7 to 21, 218, and 222 to 224; and HVR-H2sequence selected from SEQ ID NOs: 26 to 49, 232, 219, and 225; and anHVR-H3 sequence selected from SEQ ID NOs: 52 to 61, 220, 221, 226, and227. In some embodiments, the VH comprises an HVR-H1 sequence selectedfrom SEQ ID NOs: 15, 218, and 222 to 224; an HVR-H2 sequence selectedfrom SEQ ID NOs: 29, 219, and 255; and an HVR-H3 sequence selected fromSEQ ID NOs: 52, 220, 221, 226, and 227. In some embodiments, the VHcomprises an HVR-H1 of SEQ ID NO: 15; an HVR-H2 sequence of SEQ ID NO:29; and an HVR-H3 sequence of SEQ ID NO: 52. In some embodiments, the VHcomprises an HVR-H1, HVR-H2, and HVR-H3 of an antibody selected fromantibody 6266 variants 1-15.

In another aspect, an anti-BACE1 antibody comprises a light chainvariable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL of ananti-BACE1 antibody of Table 1. In another aspect, an anti-BACE1antibody is provided, wherein the antibody comprises a light chainvariable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to an amino acid sequenceselected from SEQ ID NOs: 148 to 178, 187 to 194, and 201 to 208. Insome embodiments, an anti-BACE1 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 156. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-BACE1 antibody comprising that sequenceretains the ability to bind to BACE1 and/or inhibit or reduce BACE1activity. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in any one of SEQ ID NOs: 148to 178, 187 to 194, and 201 to 208. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-BACE1 antibody comprisesthe VL sequence in any one of SEQ ID NOs: 148 to 178, 187 to 194, and201 to 208, including post-translational modifications of that sequence.In a particular embodiment, the VL comprises one, two or three HVRs ofan anti-BACE1 antibody listed in Table 1. In some embodiments, the VLcomprises an HVR-L1 sequence selected from SEQ ID NOs: 62 and 63; anHVR-L2 sequence selected from SEQ ID NOs: 69 and 70; and an HVR-L3sequence selected from SEQ ID NOs: 75 to 78 and 98. In some embodiments,the VL comprises an HVR-L1 sequence selected from SEQ ID NOs: 64 to 68;an HVR-L2 sequence selected from SEQ ID NOs: 69 to 74 and 217; and anHVR-L3 sequence selected from SEQ ID NOs: 79 to 97. In some embodiments,the VL comprises an HVR-L1 sequence of SEQ ID NO: 65; an HVR-L2 sequenceof SEQ ID NO: 71, 73, or 217; and an HVR-L3 sequence of SEQ ID NO: 80.In some embodiments, the VL comprises an HVR-L1 sequence of SEQ ID NO:65; an HVR-L2 sequence of SEQ ID NO: 71; and an HVR-L3 sequence of SEQID NO: 80. In some embodiments, the VL comprises an HVR-L1, HVR-L2, andHVR-L3 of an antibody selected from antibody 6266 variants 1-15.

In another aspect, an anti-BACE1 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above. In some embodiments,an anti-BACE1 antibody is provided, wherein the antibody comprises a VHsequence selected from SEQ ID NOs: 99 to 106 and a VL sequence selectedfrom SEQ ID NOs: 148 to 152. In some embodiments, an anti-BACE1 antibodyis provided, wherein the antibody comprises a VH sequence selected fromSEQ ID NOs: 107 to 147, 194 to 200, and 209 to 216, and a VL sequenceselected from SEQ ID NOs: 153 to 178, 187 to 194, and 201 to 208. Insome embodiments, an anti-BACE1 antibody is provided, wherein theantibody comprises a VH and a VL of an anti-BACE1 antibody listed inTable 1. In some embodiments, the antibody comprises a VH sequenceselected from SEQ ID NOs: 138, 194 to 200, and 209 to 216, and a VLsequence selected from SEQ ID NO: 156, 187 to 194, and 201 to 208,including post-translational modifications of those sequences. In someembodiments, the antibody comprises the VH and VL sequences of SEQ IDNO: 138 and SEQ ID NO: 156, respectively, including post-translationalmodifications of those sequences. In some embodiments, an anti-BACE1antibody is provided, wherein the antibody comprises a VH and a VL of anantibody selected from antibody 6266 variants 1-15.

In a further aspect, the invention provides an antibody that binds tothe same epitope as an anti-BACE1 antibody provided herein, such as theanti-BACE1 antibodies listed in Table 1. For example, in certainembodiments, an antibody is provided that binds to the same epitope asan anti-BACE1 antibody comprising a VH sequence selected from SEQ IDNOs: 99 to 106 and a VL sequence selected from SEQ ID NOs: 148 to 152.In certain embodiments, an antibody is provided that binds to the sameepitope as an anti-BACE1 antibody comprising a VH sequence selected fromSEQ ID NOs: 107 to 147, 194 to 200, and 209 to 216, and a VL sequenceselected from SEQ ID NOs: 153 to 178, 187 to 194, and 201 to 208. Incertain embodiments, an antibody is provided that binds to the sameepitope as an anti-BACE1 antibody comprising the VH and VL sequences ofSEQ ID NO: 138 and SEQ ID NO: 156, respectively.

In another embodiment, an antibody is provided that competes for binding(e.g., binds to the same epitope) as any anti-BACE1 antibody describedherein.

In a further aspect of the invention, an anti-BACE1 antibody accordingto any of the above embodiments is a monoclonal antibody, including achimeric or human antibody. In some embodiments, an anti-BACE1 antibodyis an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody, orF(ab′)₂ fragment. In another embodiment, the antibody is a full lengthantibody, e.g., an intact IgG1 antibody or other antibody class orisotype as defined herein.

In a further aspect, an anti-BACE1 antibody according to any of theabove embodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from10⁻⁹ M to 10⁻¹³ M).

In some embodiments, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881(1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CMS chips at˜10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CMS, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g., Chenet al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶M⁻s⁻¹ by the surface plasmon resonance assay above, then the on-rate canbe determined by using a fluorescent quenching technique that measuresthe increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with astirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for BACE1 and the other is for any other antigen. Incertain embodiments, bispecific antibodies may bind to two differentepitopes of BACE1. Bispecific antibodies may also be used to localizecytotoxic agents to cells which express BACE1. Bispecific antibodies canbe prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tuft et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to BACE1 as well asanother, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 2 under the heading of “conservative substitutions.” Moresubstantial changes are provided in Table 2 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 2 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanantibody). Generally, the resulting variant(s) selected for furtherstudy will have modifications (e.g., improvements) in certain biologicalproperties (e.g., increased affinity, reduced immunogenicity) relativeto the parent antibody and/or will have substantially retained certainbiological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) Insome embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized. HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as Arg, Asp, His, Lys, and Glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In some embodiments, antibody variants are provided having acarbohydrate structure that lacks fucose attached (directly orindirectly) to an Fc region. For example, the amount of fucose in suchantibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from20% to 40%. The amount of fucose is determined by calculating theaverage amount of fucose within the sugar chain at Asn297, relative tothe sum of all glycostructures attached to Asn 297 (e.g. complex, hybridand high mannose structures) as measured by MALDI-TOF mass spectrometry,as described in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Natl. Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Natl. Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model such as that disclosed inClynes et al. Proc. Natl. Acad. Sci. USA 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. See, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Intl. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) Clq binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In some embodiments, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In some embodiments,isolated nucleic acid encoding an anti-BACE1 antibody described hereinis provided. Such nucleic acid may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such nucleic acid are provided. In a further embodiment, ahost cell comprising such nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the VL of the antibody and an amino acid sequence comprisingthe VH of the antibody, or (2) a first vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VL of the antibodyand a second vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In some embodiments, thehost cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell orlymphoid cell (e.g., Y0, NS0, Sp20 cell). In some embodiments, a methodof making an anti-BACE1 antibody is provided, wherein the methodcomprises culturing a host cell comprising a nucleic acid encoding theantibody, as provided above, under conditions suitable for expression ofthe antibody, and optionally recovering the antibody from the host cell(or host cell culture medium).

For recombinant production of an anti-BACE1 antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

C. Assays

Anti-BACE1 antibodies provided herein may be identified, screened for,or characterized for their physical/chemical properties and/orbiological activities by various assays known in the art.

1. Binding Assays and Other Assays

In some aspects, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc.

In another aspect, competition assays may be used to identify anantibody that competes with any of the anti-BACE1 antibodies describedherein for binding to BACE1. In certain embodiments, such a competingantibody binds to the same epitope (e.g., a linear or a conformationalepitope) that is bound by any of the antibodies descried herein.Detailed exemplary methods for mapping an epitope to which an antibodybinds are provided in Morris (1996) “Epitope Mapping Protocols,” inMethods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized BACE1 is incubated in asolution comprising a first labeled antibody that binds to BACE1 (e.g.,an anti-BACE1 antibody described herein) and a second unlabeled antibodythat is being tested for its ability to compete with the first antibodyfor binding to BACE1. The second antibody may be present in a hybridomasupernatant. As a control, immobilized BACE1 is incubated in a solutioncomprising the first labeled antibody but not the second unlabeledantibody. After incubation under conditions permissive for binding ofthe first antibody to BACE1, excess unbound antibody is removed, and theamount of label associated with immobilized BACE1 is measured. If theamount of label associated with immobilized BACE1 is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antibody is competing with the first antibodyfor binding to BACE1. See Harlow and Lane (1988) Antibodies: ALaboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.).

2. Activity Assays

In some aspects, assays are provided for identifying anti-BACE1antibodies thereof having biological activity. Biological activity mayinclude, e.g., inhibition or reduction of BACE1 aspartyl proteaseactivity; or inhibition or reduction in APP cleavage by BACE1; orinhibition or reduction in Aβ production. Antibodies having suchbiological activity in vivo and/or in vitro are also provided.

In certain embodiments, an antibody of the invention is tested for suchbiological activity. For example, BACE1 protease activity can be testedin a homogeneous time-resolved fluorescence HTRF assay, as described indetail in the Examples, using synthetic substrate peptides.

Briefly, a homogeneous time-resolved fluorescence (HTRF) assay can beused to measure BACE1 aspartyl protease activity with the use of anamyloid precursor protein BACE1 cleavage site peptide. For example, theBi27 peptide (Biotin-KTEEISEVNLDAEFRHDSGYEVHHQKL (SEQ ID NO: 183),American Peptide Company)), is combined with BACE1 pre-incubated with ananti-BACE antibody in BACE reaction buffer (50 mM sodium acetate pH 4.4and 0.1% CHAPS) in a 384-well plate (Proxiplate™, Perkin-Elmer). Theproteolytic reaction mixture is incubated at ambient temperature for 75minutes and was quenched by the addition of 5 μL HTRF detection mixturecontaining 2 nM Streptavidin-D2 and 150 nM of an anti-amyloid betaantibody labeled with Europium cryptate in detection buffer (200 mM TrispH 8.0, 20 mM EDTA, 0.1% BSA, and 0.8M KF). The final reaction mixtureis incubated at ambient temperature for 60 minutes and the TR-FRETsignal is measured using an EnVision Multilabel Plate Reader™(Perkin-Elmer) at an excitation wavelength of 320 nm and emissionwavelengths of 615 and 665 nm.

In some embodiments, BACE1 protease activity may be measured using amicrofluidic capillary electrophoretic (MCE) assay. An MCE assayreaction can be carried out in a standard enzymatic reaction, initiatedby the addition of substrate to enzyme and 4× compound, containing humanBACE1 (extracellular domain), amyloid precursor protein beta secretaseactive site peptide (FAM-KTEEISEVNLDAEFRWKK-CONH₂ (SEQ ID NO:186)), 50mM NaOAc pH 4.4 and 0.1% CHAPS. After incubation for 60 minutes atambient temperature, the product and substrate in each reaction isseparated using a 12-sipper microfluidic chip analyzed on an LC30000(both, Caliper Life Sciences). The separation of product and substrateis optimized by choosing voltages and pressure using the manufacturer'soptimization software. Substrate conversion is calculated from theelectrophoregram using HTS Well Analyzer software (Caliper LifeSciences).

In addition, BACE1 protease activity can be tested in vivo in cell lineswhich express BACE1 substrates such as APP, as described in the Examplesherein; or in transgenic mice which express BACE1 substrates, such ashuman APP, as described in PCT Publication No. WO 2012/064836 A1.

Additionally, BACE1 protease activity can be tested with anti-BACE1antibodies in animal models. For example, animal models of variousneurological diseases and disorders, and associated techniques forexamining the pathological processes associated with these models, arereadily available in the art. Animal models of various neurologicaldisorders include both non-recombinant and recombinant (transgenic)animals. Non-recombinant animal models include, for example, rodent,e.g., murine models. Such models can be generated by introducing cellsinto syngeneic mice using standard techniques, e.g. subcutaneousinjection, tail vein injection, spleen implantation, intraperitonealimplantation, and implantation under the renal capsule. In vivo modelsinclude models of stroke/cerebral ischemia, in vivo models ofneurodegenerative diseases, such as mouse models of Parkinson's disease;mouse models of Alzheimer's disease; mouse models of amyotrophic lateralsclerosis; mouse models of spinal muscular atrophy; mouse/rat models offocal and global cerebral ischemia, for instance, common carotid arteryocclusion or middle cerebral artery occlusion models; or in ex vivowhole embryo cultures. As one nonlimiting example, there are a number ofart-known mouse models for Alzheimer's disease ((see, e.g. Rakover etal., Neurodegener. Dis. (2007); 4(5): 392-402; Mouri et al., FASEB J.(2007) July; 21 (9): 2135-48; Minkeviciene et al., J. Pharmacol. Exp.Ther. (2004) November; 311 (2):677-82 and Yuede et al., Behav Pharmacol.(2007) September; 18 (5-6): 347-63). The various assays may be conductedin known in vitro or in vivo assay formats, as known in the art anddescribed in the literature. Various such animal models are alsoavailable from commercial vendors such as the Jackson Laboratory.

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-BACE1antibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In some embodiments, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med. Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med.Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or I123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-BACE1 antibodies provided hereinis useful for detecting the presence of BACE1 in a biological sample.The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell or tissue, such as serum, plasma, saliva, gastricsecretions, mucus, cerebrospinal fluid, lymphatic fluid, neuronaltissue, brain tissue, cardiac tissue or vascular tissue.

In some embodiments, an anti-BACE1 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of BACE1 in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-BACE1 antibody as described herein under conditionspermissive for binding of the anti-BACE1 antibody to BACE1, anddetecting whether a complex is formed between the anti-BACE1 antibodyand BACE1. Such method may be an in vitro or in vivo method. In someembodiments, an anti-BACE1 antibody is used to select subjects eligiblefor therapy with an anti-BACE1 antibody, e.g. where BACE1 is a biomarkerfor selection of patients.

Exemplary disorders that may be diagnosed using an antibody of theinvention include neurodegenerative diseases (including, but not limitedto, Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome,olivopontocerebellar atrophy, Parkinson's disease, multiple systematrophy, striatonigral degeneration, tauopathies (including, but notlimited to, Alzheimer disease and supranuclear palsy), prion diseases(including, but not limited to, bovine spongiform encephalopathy,scrapie, Creutzfeldt-Jakob syndrome, kuru,Gerstmann-Straussler-Scheinker disease, chronic wasting disease, andfatal familial insomnia), stroke, muscular dystrophy, multiplesclerosis, Amyotrophic lateral sclerosis (ALS), Angelman's syndrome,Liddle syndrome, Paget's syndrome, traumatic brain injury, bulbar palsy,motor neuron disease, and nervous system heterodegenerative disorders(including, but not limited to, Canavan disease, Huntington's disease,neuronal ceroid-lipofuscinosis, Alexander's disease, Tourette'ssyndrome, Menkes kinky hair syndrome, Cockayne syndrome,Halervorden-Spatz syndrome, lafora disease, Rett syndrome,hepatolenticular degeneration, Lesch-Nyhan syndrome, andUnverricht-Lundborg syndrome), dementia (including, but not limited to,Pick's disease, and spinocerebellar ataxia).

In certain embodiments, labeled anti-BACE1 antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-BACE1 antibody as describedherein are prepared by mixing such antibody having the desired degree ofpurity with one or more optional pharmaceutically acceptable carriers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude insterstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In some aspects, a sHASEGP is combinedwith one or more additional glycosaminoglycanases such aschondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-BACE1 antibodies provided herein may be used intherapeutic methods.

In some aspects, an anti-BACE1 antibody for use as a medicament isprovided. In further aspects, an anti-BACE1 antibody for use in treatinga neurological disease or disorder is provided (e.g., AD). In certainembodiments, an anti-BACE1 antibody for use in a method of treatment isprovided. In certain embodiments, the invention provides an anti-BACE1antibody for use in a method of treating an individual having aneurological disease or disorder comprising administering to theindividual an effective amount of the anti-BACE1 antibody. In one suchembodiment, the method further comprises administering to the individualan effective amount of at least one additional therapeutic agent. Infurther embodiments, the invention provides an anti-BACE1 antibody foruse in reducing or inhibiting amlyoid plaque formation in a patient atrisk or suffering from a neurological disease or disorder (e.g., AD). Incertain embodiments, the invention provides an anti-BACE1 antibody foruse in a method of reducing or inhibiting Aβ production in an individualcomprising administering to the individual an effective of theanti-BACE1 antibody. An “individual” according to any of the aboveembodiments is preferably a human. In certain aspect, the anti-BACEantibody for use in the methods of the invention reduces or inhibitsBACE1 activity. For example, the anti-BACE1 antibody reduces or inhibitsthe ability of BACE1 to cleave APP.

In a further aspect, the invention provides for the use of an anti-BACE1antibody in the manufacture or preparation of a medicament. In someembodiments, the medicament is for treatment of neurological disease ordisorder. In a further embodiment, the medicament is for use in a methodof treating neurological disease or disorder comprising administering toan individual having neurological disease or disorder an effectiveamount of the medicament. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, e.g., as described below. In afurther embodiment, the medicament is for inhibiting BACE1 activity. Ina further embodiment, the medicament is for use in a method ofinhibiting Aβ production or plaque formation in an individual comprisingadministering to the individual an amount effective of the medicament toinhibit Aβ production or plaque formation. An “individual” according toany of the above embodiments may be a human.

In a further aspect, the invention provides a method for treatingAlzheimer's disease. In some embodiments, the method comprisesadministering to an individual having AD an effective amount of ananti-BACE1 antibody. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent. An “individual” according to anyof the above embodiments may be a human.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-BACE1 antibodies provided herein, e.g., foruse in any of the above therapeutic methods. In some embodiments, apharmaceutical formulation comprises any of the anti-BACE1 antibodiesprovided herein and a pharmaceutically acceptable carrier. In anotherembodiment, a pharmaceutical formulation comprises any of the anti-BACE1antibodies provided herein and at least one additional therapeuticagent, e.g., as described below.

Antibodies of the invention can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. Antibodies of the invention can alsobe used in combination with radiation therapy.

An antibody of the invention (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Certain embodiments of the invention provide for the antibody orfragment thereof to traverse the blood-brain barrier. Certainneurodegenerative diseases are associated with an increase inpermeability of the blood-brain barrier, such that the antibody oractive fragment thereof can be readily introduced to the brain. When theblood-brain barrier remains intact, several art-known approaches existfor transporting molecules across it, including, but not limited to,physical methods, lipid-based methods, and receptor and channel-basedmethods.

Physical methods of transporting the antibody or fragment thereof acrossthe blood-brain barrier include, but are not limited to, circumventingthe blood-brain barrier entirely, or by creating openings in theblood-brain barrier. Circumvention methods include, but are not limitedto, direct injection into the brain (see e.g., Papanastassiou et al.,Gene Therapy 9: 398-406 (2002)) and implanting a delivery device in thebrain (see e.g., Gill et al., Nature Med. 9: 589-595 (2003); and GliadelWafers™, Guildford Pharmaceutical). Methods of creating openings in thebarrier include, but are not limited to, ultrasound (see e.g., U.S.Patent Publication No. 2002/0038086), osmotic pressure (e.g., byadministration of hypertonic mannitol (Neuwelt, E. A., Implication ofthe Blood-Brain Barrier and its Manipulation, Vols 1 & 2, Plenum Press,N.Y. (1989))), permeabilization by, e.g., bradykinin or permeabilizerA-7 (see e.g., U.S. Pat. Nos. 5,112,596, 5,268,164, 5,506,206, and5,686,416), and transfection of neurons that straddle the blood-brainbarrier with vectors containing genes encoding the antibody or fragmentthereof (see e.g., U.S. Patent Publication No. 2003/0083299).

Lipid-based methods of transporting the antibody or fragment thereofacross the blood-brain barrier include, but are not limited to,encapsulating the antibody or fragment thereof in liposomes that arecoupled to antibody binding fragments that bind to receptors on thevascular endothelium of the blood-brain barrier (see e.g., U.S. PatentApplication Publication No. 20020025313), and coating the antibody oractive fragment thereof in low-density lipoprotein particles (see e.g.,U.S. Patent Application Publication No. 20040204354) or apolipoprotein E(see e.g., U.S. Patent Application Publication No. 20040131692).

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. However, other dosage regimens may be useful.The progress of this therapy is easily monitored by conventionaltechniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-BACE1 antibody.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an anti-BACE1 antibody.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1: Generation of Anti-BACE1 Antibodies

Fully-human antibodies specifically binding to BACE1 were generatedusing a yeast-based human antibody display library, selected againsthuman BACE1 extracellular domain (BACE1-ECD), amino acids 1-457 of SEQID NO: 179.

Eight naïve human synthetic yeast libraries each of ˜10⁹ diversity werepropagated as described previously (see, e.g., Xu et al, 2013, ProteinEng. Des. Sel., 26: 663-70; WO2009036379; WO2010105256; WO2012009568).For the first two rounds of selection, a magnetic bead sorting techniqueutilizing the Miltenyi MACs system was performed, as described (see,e.g., Siegel et al., 2004, J. Immunol. Methods, 286: 141-53). Briefly,yeast cells (˜10¹⁰ cells/library) were incubated with 200 nMbiotinylated BACE1-ECD for 15 min at room temperature in FACS washbuffer (phosphate-buffered saline (PBS)/0.1% bovine serum albumin(BSA)). After washing once with 50 ml ice-cold wash buffer, the cellpellet was resuspended in 40 mL wash buffer, and Streptavidin MicroBeads(500 μl) were added to the yeast and incubated for 15 min at 4° C. Next,the yeast were pelleted, resuspended in 5 mL wash buffer, and loadedonto a Miltenyi LS column. After the 5 mL was loaded, the column waswashed 3 times with 3 ml FACS wash buffer. The column was then removedfrom the magnetic field, and the yeast were eluted with 5 mL of growthmedia and then grown overnight. The following rounds of sorting wereperformed using flow cytometry. Approximately 1×10⁸ yeast were pelleted,washed three times with wash buffer, and incubated with decreasingconcentrations of biotinylated BACE1-ECD (100 to 1 nM) under equilibriumconditions at room temperature. Yeast were then washed twice and stainedwith LC-FITC (diluted 1:100) and either SA-633 (diluted 1:500) or EA-PE(diluted 1:50) secondary reagents for 15 min at 4° C. After washingtwice with ice-cold wash buffer, the cell pellets were resuspended in0.4 mL wash buffer and transferred to strainer-capped sort tubes.Sorting was performed using a FACS ARIA sorter (BD Biosciences) and sortgates were determined to select for binders. After the final round ofsorting, yeast were plated and individual colonies were picked forcharacterization.

Optimization of select antibodies was carried out by a performing bothlight chain and heavy chain diversification, each described below.

Light chain diversification: Heavy chain plasmids were extracted andtransformed into a light chain library with a diversity of approximately1×10⁶. Selections were performed as described above with one round ofMACS sorting and two rounds of FACS sorting using biotinylated BACE1-ECDtitrations so select for higher affinity.

Heavy chain diversification: The CDRH3 of was recombined into a premadelibrary with CDRH1 and CDRH2 variants of a diversity of 1×10⁸ andselections were performed as described above. Affinity pressures wereapplied by incubating the antigen antibody yeast complex with parentalFab or unbiotinylated antigen for different amounts of time to selectfor the highest affinity antibodies. Additional cycles ofdiversification utliized error prone PCR-based mutagenesis of the heavychain and the light chain. Selections were performed similar to previouscycles using FACS sorting for all three rounds and with increased timesfor Fab pressure.

Seventy-eight antibodies were chosen from the selection process forsequencing and further characterization. The heavy chain and light chainHVRs for the seventy-eight antibodies are shown in FIGS. 1 and 2,respectively. The heavy chain and light chain variable region sequencesare shown in FIGS. 3 and 4, respectively.

The antibodies were determined to be in two separate epitope bins. Theepitope bin for each antibody (either bin “2” or bin “3”) is shown inFIGS. 1 to 4.

Example 2: Characterization of Anti-BACE1 Antibodies

The anti-BACE1 antibodies selected in Example 1 were furthercharacterized using the assays described below.

A. Binding Kinetics Using Octet® System

The binding affinities for the 78 anti-BACE1 antibodies selected inExample 1 for human and murine BACE1 ECD were determined using an Octet®System (ForteBio) as follows. Anti-BACE1 antibodies were loaded ontoanti-human capture (AHC) sensors (tips) followed by 60 seconds baselinein assay buffer. Tips were then exposed to 200 nM of human BACE1 ECD(produced in CHO cells or purchased from R&D Systems) or murine BACE1ECD (produced in CHO cells; SEQ ID NO: 231). Tips were transferred toassay buffer for 5 minutes, 30 minutes, or 120 minutes depending on theoff-rate, for off-rate measurement. Assay buffer was either PBS+0.1%BSA, pH 7.5; or PBS+0.1% BSA, pH 5.0. Kinetics were analyzed using a 1:1binding model.

The results of that experiment are shown in FIG. 5. Measurements withthe “<” designations prior to the KD reach the lower limit of measurableoff-rate for that assay run.

B. Binding Kinetics Using Surface Plasmon Resonance (BIAcore™)

Binding affinities of certain anti-BACE1 IgGs were measured by surfaceplasmon resonance (SRP) using a BIAcore™-T100 instrument. Anti-BACE1human IgGs were captured by mouse anti-human Fc antibody (GE Healthcare,cat # BR-1008-39) coated on CMS biosensor chips to achieve approximately150 response units (RU). For kinetics measurements, two-fold serialdilutions (125 nM to 0 nM) of human BACE1 (R&D Systems) were injected inHBS-P buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% v/v SurfactantP20, GE Healthcare) at 25° C. with a flow rate of 30 μl/min. Associationrates (k_(on)) and dissociation rates (k_(off)) were calculated using asimple one-to-one Langmuir binding model (BIAcore Evaluation Softwareversion 3.2). The equilibrium dissociation constant (K_(D)) wascalculated as the ratio k_(off)/k_(on).

The results of that experiment are shown in FIG. 5.

C. Epitope Binning

Epitope binning of the 78 antibodies was performed using the Octet®System. Briefly, a first antibody is loaded onto multiple AHC tipsfollowed by 60 seconds baseline in assay buffer, pH 7.5. Tips are thenexposed to 200 nM human BACE1 for 180 seconds to allow for antigenbinding. Tips are transferred to wells containing 50 μg/ml secondantibody in assay buffer for 90 seconds. If the second antibody showsclear binding, it is considered to be a non-competitor (i.e., in adifferent epitope bin from the first antibody). If the second antibodydoes not show clear binding, it is considered to be a competitor (i.e.,in the same epitope bin as the first antibody). The bindingdetermination is made by comparing the second antibody binding to BACE1in the presence of the first antibody to the first antibody blockingitself. To choose the first antibodies, an assay similar to the assaydescribed above is carried out, and antibodies showing mutuallyexclusive binding are selected.

The epitope bin for each antibody is shown in FIGS. 1 to 4. The 78antibodies fell into two epitope bins.

D. In Vitro Inhibition Assays

Additionally, the ability of antibodies to modulate BACE1 proteolyticactivity on certain BACE substrates was assessed in vitro using the HTRFassay.

In the first assay, certain anti-BACE1 antibodies were diluted inreaction buffer (50 mM NaAcetate pH 4.4, 0.1% CHAPS) to approximately0.6 μM. BACE1 ECD was diluted in reaction buffer to 0.3 μM. 3 μL ofBACE1 and 3 μL of anti-BACE1 antibody were combined in a well of a 384well plate and incubated for 30 minutes. 3 μL of FRET short substrateMca-SEVNLDAEFRK(Dnp)RR-NH₂ (Mca: (7-Methoxycoumarin-4-yl)acetyl, Dnp:2,4-Dinitrophenyl; R&D Systems) (SEQ ID NO: 184) was added and mixed bypulse spin. Fluorescence of the cleaved substrate was monitored every 10minutes for 1 hour (excitation 320 nm, emission 405 nm). Each anti-BACE1antibody was tested in quadruplicate. Modulation of BACE1 activity wascalculated as ((free enzyme activity)−(IgG:enxyme activity))/(freeenzyme activity). Positive values represent inhibition and negativevalues represent activation.

The results of that experiment are shown in FIG. 6. In this shortsubstrate assay, both enzyme inhibition (positive percentages), andenzyme activation (negative values) were observed.

In the second assay, certain anti-BACE1 antibodies were tested in a HTRFassay as follows. Two microliters of 375 nM Bi27(Biotin-KTEEISEVNLDAEFRHDSGYEVHHQKL (SEQ ID NO:183), American PeptideCompany)), an amyloid precursor protein BACE1 cleavage site peptidebearing a substitution to increase sensitivity to BACE1 cleavage, wascombined with 3 μL of 125 nM BACE1 pre-incubated with an anti-BACEantibody in BACE reaction buffer (50 mM sodium acetate pH 4.4 and 0.1%CHAPS) in a 384-well plate (Proxiplate™, Perkin-Elmer). The proteolyticreaction mixture was incubated at ambient temperature for 75 minutes andwas quenched by the addition of 5 μL HTRF detection mixture containing 2nM Streptavidin-D2 and 150 nM of 6E10 anti-amyloid beta antibody(Covance, Emoryville, Calif.) labeled with Europium cryptate indetection buffer (200 mM Tris pH 8.0, 20 mM EDTA, 0.1% BSA, and 0.8MKF). The final reaction mixture was incubated at ambient temperature for60 minutes and the TR-FRET signal was measured using an EnVisionMultilabel Plate Reader™ (Perkin-Elmer) at an excitation wavelength of320 nm and emission wavelengths of 615 and 665 nm. Reactions lackingBACE1 enzyme and reactions lacking anti-BACE1 antibodies were used ascontrols. Additionally, reactions using a short FRET peptide(Rh-EVNLDAEFK-quencher (SEQ ID NO: 185), Invitrogen) were also performedidentically to the HTRF reactions described above.

A synthetic peptide inhibitor of BACE1, OM99-2 (CalBiochem®, Catalog#496000) was used as a control. The resulting fluorogenic products fromthe control reactions were measured as above, but at an excitationwavelength of 545 nm and an emission wavelength of 585 nm. Obtained datawere analyzed using GraphPad Prism 5™ (LaJolla, Calif.).

The results of that experiment are shown in FIG. 7. A “decreasing” datamode indicates inhibition, while an “increasing” data mode indicatedactivation.

Without intending to be bound by any particular theory, modulation ofenzyme activity by allosteric inhibitors may, in some instances, resultin either activation or inhibition of activity depending on thesubstrate. For example, a change in conformation caused by an allostericinhibitor may cause better binding of one substrate and may interferewith binding of another substrate, resulting in differing, or evenopposite, activity modulation.

E. In Vivo Activity Assay in Primary Cultures

To determine whether the observed in vitro inhibitory action of theanti-BACE1 antibodies on APP processing was also present in a cellularcontext, in vivo studies were performed. The ability of the antibodiesto inhibit Apx-40 production in primary cultures of mouse corticalneurons expressing endogenous levels of wild-type human amyloidprecursor protein was assessed as follows. Briefly, dissociated corticalneuron cultures were prepared from E16.5 CD1 mice. Neurons were seededat a density of 2.5×10⁴ cells/well in a 96-well plate and grown for fivedays in Neurobasal media (Life Technologies) in vitro. 50 μl of freshmedia containing anti-BACE antibodies or control IgG1 prepared in an8-point dilution series was incubated with the neurons for 24 hours at37° C. Cell supernatants were harvested and assayed for the presence ofmouse Aβ_(x-40) using a sandwich ELISA Briefly, rabbit polyclonalantibody specific for the C terminus of Aβ_(x-40) (Millipore, Bedford,Mass.) was coated onto plates, and biotinylated anti-mouse Aβ monoclonalantibody M3.2 (Covance, Dedham, Mass.) was used for detection. The assayhad lower limit of quantification values of 1.96 pg/ml in plasma and39.1 pg/g in brain. Aβ_(x-40) values were normalized for cell viability,as determined using the CellTiter-Glo Luminescent Cell Viability Assay(Promega). Data was plotted using a four-parameter non-linear regressioncurve-fitting program (Prism, Graphpad).

The results of that experiment are shown in FIG. 8. Percent inhibitionrefers to the maximum inhibition (as % of control) seen with eachantibody; percent inhibition was determined as follows: (baselineAβ_(x-40)−minimal Aβ_(x-40))/baseline Aβ_(x-40)*100 (baseline Aβ_(x-40)is in the absence of any treatment).

A similar experiment was performed with antibody 6266 and YW412.8.31.

The results of that experiment are shown in FIG. 9. Antibody 6266 had anIC₅₀ of 1.7 nM and a maximum inhibition of 79%, compared to an IC₅₀ of2.9 nM and a maximum inhibition of 62% exhibited by antibody YW412.8.31(see PCT Publication No. WO 2012/064836 A1).

F. In Vivo Activity Assay in Mice

The ability of anti-BACE1 antibodies to modulate amyloidogenicprocessing was also assessed in wild-type mice. A single dose of controlIgG antibody or an anti-BACE1 antibody (100 mg/kg) was deliveredsystemically by intraperitoneal (IP) injection to 8-week old wild-typeC57B1/6J mice (n=6 per group). After 24 hours, brain samples wereharvested following PBS perfusion, and forebrain from one hemibrain washomogenized in 5M GuHCL, 50 mM Tris pH 8.0, and further diluted inCasein Blocking Buffer (0.25% casein/0.05% sodium azide, 20 μg/mlaprotinin/5 mM EDTA, pH 8.0/10 μg/ml leupeptin in PBS) for Aβ_(x-40)analysis. The concentrations of total mouse Aβ_(x-40) in brain weredetermined using a sandwich ELISA. Briefly, rabbit polyclonal antibodyspecific for the C terminus of Aβ₁₋₄₀ (Millipore, Bedford, Mass.) wascoated onto plates, and biotinylated anti-mouse Aβ monoclonal antibodyM3.2 (Covance, Dedham, Mass.) was used for detection. The assay hadlower limit of quantification values of 1.96 pg/ml in plasma and 39.1pg/g in brain.

The results of that experiment are shown in FIG. 10. Administration ofantibody 5884 showed the greatest reduction in Aβ_(x-40) levels in mousebrain. Antibody 6226 is derived from antibody 5884, and varies at onlytwo positions in the heavy chain (in HVR-H1).

G. Pharmacokinetics in Cynomolgus Monkeys

The PK profiles of antibody 6266 and antibody 6310 were compared to thePK profile of antibody YW412.8.31 (see PCT Publication No. WO2012/064836 A1). Antibodies were administered as a single intravenous(IV) dose at 10 mg/kg. Each antibody was administered to four monkeys.Antibody concentration in serum was measured at the following timepoints: 7 days pre-dose, 15 minutes and 8 hours post-dose, and 1, 3, 7,10, 14, 17, 21, 28, 35, and 42 days post-dose. The concentrations of thedosed antibodies in cynomolgus monkey serum were measured with an ELISAusing a sheep anti-human IgG monkey adsorbed antibody coat, followed byadding serum samples starting at a dilution of 1:100, and finished byadding a goat anti-human IgG antibody conjugated to horseradishperoxidase monkey adsorbed for detection. The assay had a standard curverange of 0.78-50 mg/mL and a limit of detection of 0.08 mg/mL. Resultsbelow this limit of detection were reported as less than reportable(LTR).

The results of that experiment are shown in FIG. 11. No significantdifference was observed in the cynomolgus monkey pharmacokinetics ofantibody 6266 compared to YW412.8.31. Antibody 6310 was clearedapproximately twice as fast as YW412.8.31.

Example 3: Affinity Maturation of 6266 Antibody

Antibody 6266 was affinity matured as follows.

NNK Walk Library Design and Phage Panning

For library randomization, each position of the CDRs was randomized byoligonucleotide-directed mutagenesis with an “NNK” codon, where N is anyof the four natural nucleotides, and K is 50% T (thymine) and 50% G(guanine). The NNK codon can encode any of the 20 natural amino acids.Libraries for the light chain and heavy chain were made separately, andeach of the 3 CDRs of each chain was randomized at the same time. Thisresults in clones that have 0 to 3 random amino acid changes in eachchain, with up to one mutation in each CDR. Libraries were made in aphage Fab fragment display vector by standard methods. Binding cloneswere selected by incubating the phage display libraries with 5, 0.5, and0.1 nM biotinylated BACE1 in successive rounds of selection, and thencompeted with 100 nM non-biotinylated BACE1 at room temperature or 37°C. to reduce binding of the lower affinity clones to BACE1. Bound cloneswere captured on ELISA plates coated with neutravidin, washed and elutedin 100 mM HCl for 20 minutes at room temperature. The eluted phage wasneutralized with 1/10 volume of 1 M Tris pH 8.0 and used to infect E.coli for amplification for the next round of selection.

Deep Sequencing and Data Analysis

For deep sequencing, phagemid DNA was isolated from selected rounds. TheVH and the VL segment from each sample were amplified by an 18 cycle PCRamplification using Phusion DNA polymerase (New England Biolabs). Theamplicon was purified on a 2% agarose gel. Amplicons were prepared withstandard Illumina library prep methods, using TruSeq DNA Sample Prep(Illumina). Adapter-ligated libraries were subjected to a single cycleof PCR and sequenced on the Illumina MiSeq, paired-end 200 bp or 300 bpas appropriate to cover the entire length of the amplicon. Sequencingdata where analyzed using the statistical programming language R and theShortRead package. Quality control was performed on identified CDRsequences, were each CDR sequence was checked for the correct length andwas allowed to carry only up to one NNK mutation and no non-NNKmutations. Calculating the frequency of all mutations of everyrandomized position generated position weight matrices. Enrichmentratios for all single mutations were calculated by dividing thefrequency of a given mutation at a given position in the sorted sampleby the frequency of the very same mutation in the unsorted sample, asdescribed previously by Fowler and colleagues. The enrichment ratios ofdouble mutations were obtained by calculating the enrichment ratio ofall clones that carry NNK mutations at two given positions, ignoring thethird NNK mutation. In order to filter out sampling effects, mutationpairs that had less than 10 sequence counts either in the sorted orunsorted sample, were removed from the analysis. Epistasis wascalculated by combining the enrichment ratios from single and doublemutation in a multiplicative model: EnrichAB=EnrichA×EnrichB. Theepistasis used is thus defined as: Epistasis=EnrichAB−EnrichA×EnrichB.The highest enriched mutation from the single mutation analysis and fromthe double mutation analysis were selected for synthesis.

FIGS. 12 and 13 show the heavy chain and light chain variable regionsequences of affinity matured antibody 6266 variants 1 to 15.

Affinity Determination Using Surface Plasmon Resonance

The binding affinity of anti-BACE1 Fab antibodies by single-cyclekinetics was determined using surface plasmon resonance (SRP)measurement with a BIAcore™ T200 instrument. Briefly, series S sensorchip CMS was activated with EDC and NHS reagents according to thesupplier's instructions, and anti-His antibody was coupled to achieveapproximately 1000 response units (RU), then following by blockingun-reacted groups with 1M ethanolamine. For kinetics measurements,His-tagged BACE1 protein was first injected at 10 μl/min flow rate tocapture approximately 100 RU at 3 different flow cells (FC), except forFC1 (reference), and then 5-fold serial dilutions of Fab in HBS-P buffer(0.01M HEPES pH 7.4, 0.15M NaCl, 0.005% surfactant P20) from low (0.08nM) to high (50 nM) were injected (flow rate: 34 μl/min) one after theother in the same cycle with no regeneration between injections. Thesensorgram was recorded and subject to reference and buffer subtractionbefore evaluating by BIAcore™ T200 Evaluation Software (version 2.0).Association rates (k_(on)) and dissociation rates (k_(off)) werecalculated using a simple one-to-one Langmuir binding model. Theequilibrium dissociation constant (Kd) was calculated as the ratiok_(off)/k_(on).

Thermal Melt Temperature (TM) Determination by Differential ScanningFluorimetry (DSF)

DSF monitors thermal unfolding of proteins in the presence of afluorescent dye and is typically performed by using a real-time PCRinstrument (e.g., Bio-Rad CFX). SYPRO orange dye (Invitrogen, cat. no.56650) is diluted 1:20 in PBS. One μl of diluted dye is added to 24 μlFab protein (˜100 ug/ml) in a well. As the temperature increases from20° C. to 100° C. in the real-time PCR instrument (Bio-Rad CFX), thefluorescence intensity is plotted and the inflection point of thetransition curve (Tm) is calculated using, for example, the Boltzmannequation. See Nature Protocols, 2007, 2:2212-2221.

FIG. 14 shows the association rate, dissociation rate, dissociationconstant, and melting temperature for the affinity matured antibody 6266variants 1 to 15. All of the variants showed improved affinity (K_(D))compared to antibody 6266.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

Table of Sequences SEQ ID NO Descripton Sequence 179 Human BACE1,MAQALPWLLL WMGAGVLPAH GTQHGIRLPL RSGLGGAPLG isoform BLRLPRETDEE PEEPGRRGSF VEMVDNLRGK SGQGYYVEMTVGSPPQTLNI LVDTGSSNFA VGAAPHPFLH RYYQRQLSSTYRDLRKGVYV PYTQGKWEGE LGTDLVSIPH GPNVTVRANIAAITESDKFF INGSNWEGIL GLAYAEIARP DDSLEPFFDSLVKQTHVPNL FSLQLCGAGF PLNQSEVLAS VGGSMIIGGIDHSLYTGSLW YTPIRREWYY EVIIVRVEIN GQDLKMDCKEYNYDKSIVDS GTTNLRLPKK VFEAAVKSIK AASSTEKFPDGFWLGEQLVC WQAGTTPWNI FPVISLYLMG EVTNQSFRITILPQQYLRPV EDVATSQDDC YKFAISQSST GTVMGAVIMEGFYVVFDRAR KRIGFAVSAC HVHDEFRTAA VEGPFVTLDMEDCGYNIPQT DESTLMTIAY VMAAICALFM LPLCLMVCQW CCLRCLRQQH DDFADDISLL K 180Human BACE1, MAQALPWLLL WMGAGVLPAH GTQHGIRLPL RSGLGGAPLG isoform ALRLPRETDEE PEEPGRRGSF VEMVDNLRGK SGQGYYVEMTVGSPPQTLNI LVDTGSSNFA VGAAPHPFLH RYYQRQLSSTYRDLRKGVYV PYTQGKWEGE LGTDLVSIPH GPNVTVRANIAAITESDKFF INGSNWEGIL GLAYAEIARL CGAGFPLNQSEVLASVGGSM IIGGIDHSLY TGSLWYTPIR REWYYEVIIVRVEINGQDLK MDCKEYNYDK SIVDSGTTNL RLPKKVFEAAVKSIKAASST EKFPDGFWLG EQLVCWQAGT TPWNIFPVISLYLMGEVTNQ SFRITILPQQ YLRPVEDVAT SQDDCYKFAISQSSTGTVMG AVIMEGFYVV FDRARKRIGF AVSACHVHDEFRTAAVEGPF VTLDMEDCGY NIPQTDESTL MTIAYVMAAICALFMLPLCL MVCQWCCLRC LRQQHDDFAD DISLLK 181 Human BACE1,MAQALPWLLL WMGAGVLPAH GTQHGIRLPL RSGLGGAPLG isoform CLRLPRETDEE PEEPGRRGSF VEMVDNLRGK SGQGYYVEMTVGSPPQTLNI LVDTGSSNFA VGAAPHPFLH RYYQRQLSSTYRDLRKGVYV PYTQGKWEGE LGTDLPDDSL EPFFDSLVKQTHVPNLFSLQ LCGAGFPLNQ SEVLASVGGS MIIGGIDHSLYTGSLWYTPI RREWYYEVII VRVEINGQDL KMDCKEYNYDKSIVDSGTTN LRLPKKVFEA AVKSIKAASS TEKFPDGFWLGEQLVCWQAG TTPWNIFPVI SLYLMGEVTN QSFRITILPQQYLRPVEDVA TSQDDCYKFA ISQSSTGTVM GAVIMEGFYVVFDRARKRIG FAVSACHVHD EFRTAAVEGP FVTLDMEDCGYNIPQTDEST LMTIAYVMAA ICALFMLPLC LMVCQWCCLR CLRQQHDDFA DDISLLK 182Human BACE1, MAQALPWLLL WMGAGVLPAH GTQHGIRLPL RSGLGGAPLG isoform DLRLPRETDEE PEEPGRRGSF VEMVDNLRGK SGQGYYVEMTVGSPPQTLNI LVDTGSSNFA VGAAPHPFLH RYYQRQLSSTYRDLRKGVYV PYTQGKWEGE LGTDLLCGAG FPLNQSEVLASVGGSMIIGG IDHSLYTGSL WYTPIRREWY YEVIIVRVEINGQDLKMDCK EYNYDKSIVD SGTTNLRLPK KVFEAAVKSIKAASSTEKFP DGFWLGEQLV CWQAGTTPWN IFPVISLYLMGEVTNQSFRI TILPQQYLRP VEDVATSQDD CYKFAISQSSTGTVMGAVIM EGFYVVFDRA RKRIGFAVSA CHVHDEFRTAAVEGPFVTLD MEDCGYNIPQ TDESTLMTIA YVMAAICALFMLPLCLMVCQ WCCLRCLRQQ HDDFADDISL LK 228 Murine BACE1,MAPALHWLLL WVGSGMLPAQ GTHLGIRLPL RSGLAGPPLG isoform 1LRLPRETDEE SEEPGRRGSF VEMVDNLRGK SGQGYYVEMTVGSPPQTLNI LVDTGSSNFA VGAAPHPFLH RYYQRQLSSTYRDLRKGVYV PYTQGKWEGE LGTDLVSIPH GPNVTVRANIAAITESDKFF INGSNWEGIL GLAYAEIARP DDSLEPFFDSLVKQTHIPNI FSLQLCGAGF PLNQTEALAS VGGSMIIGGIDHSLYTGSLW YTPIRREWYY EVIIVRVEIN GQDLKMDCKEYNYDKSIVDS GTTNLRLPKK VFEAAVKSIK AASSTEKFPDGFWLGEQLVC WQAGTTPWNI FPVISLYLMG EVTNQSFRITILPQQYLRPV EDVATSQDDC YKFAVSQSST GTVMGAVIMEGFYVVFDRAR KRIGFAVSAC HVHDEFRTAA VEGPFVTADMEDCGYNIPQT DESTLMTIAY VMAAICALFM LPLCLMVCQW RCLRCLRHQH DDFADDISLL K 229Murine BACE1, MAPALHWLLL WVGSGMLPAQ GTHLGIRLPL RSGLAGPPLG isoform 2LRLPRETDEE SEEPGRRGSF VEMVDNLRGK SGQGYYVEMTVGSPPQTLNI LVDTGSSNFA VGAAPHPFLH RYYQRQLSSTYRDLRKGVYV PYTQGKWEGE LGTDLVSIPH GPNVTVRANIAAITESDKFF INGSNWEGIL GLAYAEIARP DDSLEPFFDSLVKQTHIPNI FSLQLCGAGF PLNQTEALAS VGGSMIIGGIDHSLYTGSLW YTPIRREWYY EVIIVRVEIN GQDLKMDCKETEKFPDGFWL GEQLVCWQAG TTPWNIFPVI SLYLMGEVTNQSFRITILPQ QYLRPVEDVA TSQDDCYKFA VSQSSTGTVMGAVIMEGFYV VFDRARKRIG FAVSACHVHD EFRTAAVEGPFVTADMEDCG YNIPQTDEST LMTIAYVMAA ICALFMLPLCLMVCQWRCLR CLRHQHDDFA DDISLLK 230 human BACE1 ECDMAQALPWLLL WMGAGVLPAH GTQHGIRLPL RSGLGGAPLGLRLPRETDEE PEEPGRRGSF VEMVDNLRGK SGQGYYVEMTVGSPPQTLNI LVDTGSSNFA VGAAPHPFLH RYYQRQLSSTYRDLRKGVYV PYTQGKWEGE LGTDLVSIPH GPNVTVRANIAAITESDKFF INGSNWEGIL GLAYAEIAR  PDDSLEPFFDSLVKQTHVPN LFSLQLCGAG FPLNQSEVLA SVGGSMIIGGIDHSLYTGSL WYTPIRREWY YEVIIVRVEI NGQDLKMDCKEYNYDKSIVD SGTTNLRLPK KVFEAAVKSI KAASSTEKFPDGFWLGEQLV CWQAGTTPWN IFPVISLYLM GEVTNQSFRITILPQQYLRP VEDVATSQDD CYKFAISQSS TGTVMGAVIMEGFYVVFDRA RKRIGFAVSA CHVHDEFRTA AVEGPFVTLD MEDCGYNIPQ TDESTLMTGR A 231Murine BACE1 ECD MAPALHWLLL WVGSGMLPAQ GTHLGIRLPL RSGLAGPPLGLRLPRETDEE SEEPGRRGSF VEMVDNLRGK SGQGYYVEMTVGSPPQTLNI LVDTGSSNFA VGAAPHPFLH RYYQRQLSSTYRDLRKGVY  VPYTQGKWEG ELGTDLVSIP HGPNVTVRANIAAITESDKF FINGSNWEGI LGLAYAEIAR PDDSLEPFFDSLVKQTHIPN IFSLQLCGAG FPLNQTEALA SVGGSMIIGGIDHSLYTGRL WYTPIRREWY YEVIIVRVEI NGQDLKMDCKEYNYDKSIVD SGTTNLRLPK KVFEAAVKSI KAASSTEKFPDGFWLGEQLV CWQAGTTPWN IFPVISLYLM GEVTNQSFRITILPQQYLRP VEDVATSQDD CYKFAVSQSS TGTVMGAVIMEGFYVVFDRA RKRIGFAVSA CHVHDEFRTA AVEGPFVTAD MEDCGYNIPQ TDESTLMTGR A 183Bi27 peptide Biotin-KTEEISEVNLDAEFRHDSGYEVHHQKL 184 FRET shortMca-SEVNLDAEFRK(Dnp)RR-NH2 substrate (R&D Systems) 185 FRET shortRh-EVNLDAEFK-quencher substrate (Invitrogen) 186 amyloid precursorFAM-KTEEISEVNLDAEFRWKK-CONH2 protein beta secretase active site peptide217 VL HVR2 of 6266.3, GASTRAY 6266.6, 6266.7, 6266.8, 6266.11 218VH HVR1 of 6266.1, GTLSHYGVS 6266.2, 6266.3, 6266.4, 6266.5,6266.6, 6266.7 219 VH HVR2 of 6266.1, NIIPGIGTANYAQKFQG 6266.2, 6266.3,6266.4, 6266.5, 6266.6, 6266.7 220 VH HVR3 for ARSGGTQYGMLDV6266.5, 6266.6 221 VH HVR3 for ARSGGTKYGELDV 6266.7, 6266.15 222VH HVR1 for GTLKGYGVS 6266.8, 6266.9 223 VH HVR1 for GTLNGYGVS 6266.12224 VH HVR1 for GTLSGYGMS 6266.13 225 VH HVR2 for NIIPGFGVANYAQKFQG6266.14 226 VH HVR3 for ARGGGTKYGMLDV 6266.8, 6266.9 227 VH HVR3 forARSGGTKWGMLDV 6266.14

What is claimed is:
 1. An isolated antibody that binds to BACE1, whereinthe antibody comprises: an HVR-H1 sequence selected from SEQ ID NOs: 15,218, and 222 to 224; an HVR-H2 sequence selected from SEQ ID NOs: 29,219, and 225; an HVR-H3 sequence selected from SEQ ID NOs: 52, 220, 221,226, and 227; the HVR-L1 sequence of SEQ ID NO: 65; an HVR-L2 sequenceselected from SEQ ID NOs: 71, 73 and 217; and the HVR-L3 sequence of SEQID NO:
 80. 2. The isolated antibody of claim 1, wherein the antibodycomprises: a) a heavy chain variable domain sequence having at least 90%sequence identity to a heavy chain variable domain sequence selectedfrom SEQ ID NOs: 138, 194 to 200, and 209 to 216; or b) a light chainvariable domain sequence having at least 90% sequence identity to alight chain variable domain sequence selected from SEQ ID NOs: 156, 187to 193, and 201 to 208; or c) a heavy chain variable domain sequence asin (a) and a light chain variable domain sequence as in (b).
 3. Theisolated antibody of claim 1, wherein the antibody comprises: a) a heavychain variable domain sequence selected from SEQ ID NOs: 138, 194 to200, and 209 to 216; or b) a light chain variable domain sequenceselected from SEQ ID NOs: 156, 187 to 193, and 201 to 208; or c) a heavychain variable domain sequence as in (a) and a light chain variabledomain sequence as in (b).
 4. The isolated antibody of claim 1, whereinthe antibody inhibits the activity of BACE1.
 5. The isolated antibody ofclaim 4, wherein the antibody inhibits BACE1 activity in a homogeneoustime-resolved fluorescence (HTRF) assay.
 6. The isolated antibody ofclaim 4, wherein the antibody inhibits BACE1 activity in a cell linethat expresses a BACE1 substrate.
 7. The isolated antibody of claim 6,wherein the BACE1 substrate is amyloid precursor protein (APP).
 8. Theisolated antibody of claim 4, wherein the antibody inhibits BACE1activity in tissue from an animal that has been administered theanti-BACE1 antibody.
 9. The isolated antibody of claim 8, wherein thetissue is brain tissue.
 10. The isolated antibody of claim 8, whereinthe animal is selected from a mouse, rat, rabbit, dog, monkey, andnon-human primate.
 11. The isolated antibody of claim 4, wherein theantibody achieves a maximum inhibition of BACE1 activity of greater than60%, as measured using the dissociated cortical neuron culture assay.12. The isolated antibody of claim 1, wherein the antibody is amonoclonal antibody.
 13. The isolated antibody of claim 12, wherein theantibody is a human antibody or a chimeric antibody.
 14. Apharmaceutical formulation comprising the antibody of claim 4 and apharmaceutically acceptable carrier.
 15. The isolated antibody of claim1, wherein the antibody binds BACE1 with an affinity (KD) of between 0.1nM and 10 nM, as measured by surface plasmon resonance (SPR).
 16. Theisolated antibody of claim 1, wherein the antibody is an antibodyfragment.
 17. The isolated antibody of claim 16, wherein the antibodyfragment is selected from a Fab, Fab′, Fab′-SH, F(ab′)2, Fv, and scFv.18. The isolated antibody of claim 1, wherein the antibody is a fulllength IgG1 antibody.
 19. An immunoconjugate comprising the antibody ofclaim 1 and a cytotoxic agent.
 20. The antibody of claim 1, wherein theantibody comprises: (a) the HVR-H1 sequence of SEQ ID NO: 15; the HVR-H2sequence of SEQ ID NO: 29; the HVR-H3 sequence of SEQ ID NO: 52; theHVR-L1 sequence of SEQ ID NO: 65; the HVR-L2 sequence of SEQ ID NO: 71;and the HVR-L3 sequence of SEQ ID NO: 80; or (b) the HVR-H1 sequence ofSEQ ID NO: 218; the HVR-H2 sequence of SEQ ID NO: 219; the HVR-H3sequence of SEQ ID NO: 52; the HVR-L1 sequence of SEQ ID NO: 65; theHVR-L2 sequence of SEQ ID NO: 71; and the HVR-L3 sequence of SEQ ID NO:80; or (c) the HVR-H1 sequence of SEQ ID NO: 218; the HVR-H2 sequence ofSEQ ID NO: 219; the HVR-H3 sequence of SEQ ID NO: 52; the HVR-L1sequence of SEQ ID NO: 65; the HVR-L2 sequence of SEQ ID NO: 73; and theHVR-L3 sequence of SEQ ID NO: 80; or (d) the HVR-H1 sequence of SEQ IDNO: 218; the HVR-H2 sequence of SEQ ID NO: 219; the HVR-H3 sequence ofSEQ ID NO: 52; the HVR-L1 sequence of SEQ ID NO: 65; the HVR-L2 sequenceof SEQ ID NO: 217; and the HVR-L3 sequence of SEQ ID NO: 80; or (e) theHVR-H1 sequence of SEQ ID NO: 218; the HVR-H2 sequence of SEQ ID NO:219; the HVR-H3 sequence of SEQ ID NO: 52; the HVR-L1 sequence of SEQ IDNO: 65; the HVR-L2 sequence of SEQ ID NO: 73; and the HVR-L3 sequence ofSEQ ID NO: 80; or (f) the HVR-H1 sequence of SEQ ID NO: 218; the HVR-H2sequence of SEQ ID NO: 219; the HVR-H3 sequence of SEQ ID NO: 220; theHVR-L1 sequence of SEQ ID NO: 65; the HVR-L2 sequence of SEQ ID NO: 73;and the HVR-L3 sequence of SEQ ID NO: 80; or (g) the HVR-H1 sequence ofSEQ ID NO: 218; the HVR-H2 sequence of SEQ ID NO: 219; the HVR-H3sequence of SEQ ID NO: 220; the HVR-L1 sequence of SEQ ID NO: 65; theHVR-L2 sequence of SEQ ID NO: 217; and the HVR-L3 sequence of SEQ ID NO:80; or (h) the HVR-H1 sequence of SEQ ID NO: 218; the HVR-H2 sequence ofSEQ ID NO: 219; the HVR-H3 sequence of SEQ ID NO: 221; the HVR-L1sequence of SEQ ID NO: 65; the HVR-L2 sequence of SEQ ID NO: 217; andthe HVR-L3 sequence of SEQ ID NO: 80; or (i) the HVR-H1 sequence of SEQID NO: 222; the HVR-H2 sequence of SEQ ID NO: 29; the HVR-H3 sequence ofSEQ ID NO: 226; the HVR-L1 sequence of SEQ ID NO: 65; the HVR-L2sequence of SEQ ID NO: 217; and the HVR-L3 sequence of SEQ ID NO: 80; or(j) the HVR-H1 sequence of SEQ ID NO: 222; the HVR-H2 sequence of SEQ IDNO: 29; the HVR-H3 sequence of SEQ ID NO: 226; the HVR-L1 sequence ofSEQ ID NO: 65; the HVR-L2 sequence of SEQ ID NO: 73; and the HVR-L3sequence of SEQ ID NO: 80; or (k) the HVR-H1 sequence of SEQ ID NO: 15;the HVR-H2 sequence of SEQ ID NO: 29; the HVR-H3 sequence of SEQ ID NO:52; the HVR-L1 sequence of SEQ ID NO: 65; the HVR-L2 sequence of SEQ IDNO: 73; and the HVR-L3 sequence of SEQ ID NO: 80; or (l) the HVR-H1sequence of SEQ ID NO: 15; the HVR-H2 sequence of SEQ ID NO: 29; theHVR-H3 sequence of SEQ ID NO: 52; the HVR-L1 sequence of SEQ ID NO: 65;the HVR-L2 sequence of SEQ ID NO: 217; and the HVR-L3 sequence of SEQ IDNO: 80; or (m) the HVR-H1 sequence of SEQ ID NO: 223; the HVR-H2sequence of SEQ ID NO: 29; the HVR-H3 sequence of SEQ ID NO: 52; theHVR-L1 sequence of SEQ ID NO: 65; the HVR-L2 sequence of SEQ ID NO: 71;and the HVR-L3 sequence of SEQ ID NO: 80; or (n) the HVR-H1 sequence ofSEQ ID NO: 224; the HVR-H2 sequence of SEQ ID NO: 29; the HVR-H3sequence of SEQ ID NO: 52; the HVR-L1 sequence of SEQ ID NO: 65; theHVR-L2 sequence of SEQ ID NO: 71; and the HVR-L3 sequence of SEQ ID NO:80; or (o) the HVR-H1 sequence of SEQ ID NO: 15; the HVR-H2 sequence ofSEQ ID NO: 225; the HVR-H3 sequence of SEQ ID NO: 227; the HVR-L1sequence of SEQ ID NO: 65; the HVR-L2 sequence of SEQ ID NO: 71; and theHVR-L3 sequence of SEQ ID NO: 80; or (p) the HVR-H1 sequence of SEQ IDNO: 15; the HVR-H2 sequence of SEQ ID NO: 29; the HVR-H3 sequence of SEQID NO: 221; the HVR-L1 sequence of SEQ ID NO: 65; the HVR-L2 sequence ofSEQ ID NO: 71; and the HVR-L3 sequence of SEQ ID NO:
 80. 21. Theantibody of claim 1, wherein the antibody comprises: (a) the heavy chainvariable domain sequence of SEQ ID NO: 138; and the light chain variabledomain sequence of SEQ ID NO: 156; or (b) the heavy chain variabledomain sequence of SEQ ID NO: 194; and the light chain variable domainsequence of SEQ ID NO: 187; or (c) the heavy chain variable domainsequence of SEQ ID NO: 195; and the light chain variable domain sequenceof SEQ ID NO: 188; or (d) the heavy chain variable domain sequence ofSEQ ID NO: 196; and the light chain variable domain sequence of SEQ IDNO: 189; or (e) the heavy chain variable domain sequence of SEQ ID NO:197; and the light chain variable domain sequence of SEQ ID NO: 190; or(f) the heavy chain variable domain sequence of SEQ ID NO: 198; and thelight chain variable domain sequence of SEQ ID NO: 191; or (g) the heavychain variable domain sequence of SEQ ID NO: 199; and the light chainvariable domain sequence of SEQ ID NO: 192; or (h) the heavy chainvariable domain sequence of SEQ ID NO: 200; and the light chain variabledomain sequence of SEQ ID NO: 193; or (i) the heavy chain variabledomain sequence of SEQ ID NO: 209; and the light chain variable domainsequence of SEQ ID NO: 201; or (j) the heavy chain variable domainsequence of SEQ ID NO: 210; and the light chain variable domain sequenceof SEQ ID NO: 202; or (k) the heavy chain variable domain sequence ofSEQ ID NO: 211; and the light chain variable domain sequence of SEQ IDNO: 203; or (l) the heavy chain variable domain sequence of SEQ ID NO:212; and the light chain variable domain sequence of SEQ ID NO: 204; or(m) the heavy chain variable domain sequence of SEQ ID NO: 213; and thelight chain variable domain sequence of SEQ ID NO: 205; or (n) the heavychain variable domain sequence of SEQ ID NO: 214; and the light chainvariable domain sequence of SEQ ID NO: 206; or (o) the heavy chainvariable domain sequence of SEQ ID NO: 215; and the light chain variabledomain sequence of SEQ ID NO: 207; or (p) the heavy chain variabledomain sequence of SEQ ID NO: 216; and the light chain variable domainsequence of SEQ ID NO: 208.